Degraded TPO agonist antibody

ABSTRACT

The invention relates to a modified antibody which contains two or more H chain V regions and two or more L chain V regions of monoclonal antibody and can transduce a signal into cells by crosslinking TPO receptor to thereby exert TPO agonist action. The modified antibody can be used as a TPO signal transduction agonist and, therefore, useful as a remedy for various diseases such as platelet-reduction-related blood diseases, thrombopenia following chemotherapy for cancer or leukemia, etc.

TECHNICAL FIELD

This invention relates to modified antibodies containing two or more Hchain V regions and two or more L chain V regions of an antibody whichshow TPO agonist activity by crosslinking TPO receptor. The modifiedantibodies have TPO agonist activity of transducing a signal into cellsby crosslinking TPO receptor and are useful as a medicine for variouspurposes.

BACKGROUND ART

Thrombopoietin (TPO) is a platelet production regulation factor found in1994 and is known to be composed of a glycoprotein having a molecularweight of 70-80 thousands produced mainly in liver. Thrombopoietin is acytokine which in bone marrow promotes platelet precursor cells tosurvive, proliferate, differentiate and mature, namely promotesmegakaryocytes to differentiate and proliferate. Thrombopoietin (TPO)receptor was identified earlier than TPO as c-Mpl, a receptor of aspecific factor to regulate platelet production (M. Souyri et al., Cell63: 1137 (1990)). It was reported that c-Mpl is distributed mainly inplatelet precursor cells, megakayocytes and platelet cells and that thesuppression of c-Mpl expression inhibits selectively megakaryocyteformation (M. Methia et al., Blood 82: 1395 (1993)). It was reportedthat the ligand to c-Mpl is TPO based on the results of proliferationassay of cells specific to c-Mpl ligand and purification of the ligandusing c-Mpl (F. de Sauvage et al., Nature 369: 533 (1994); TD. Bartleyet al., Cell 77: 1117 (1994)). At present Mpl is called TPO receptor.Therefore TPO and TPO receptor agonists have been expected to work as atherapeutic agent for thrombocytopenia, for example, as a medicinealleviating thrombocytopenia caused by bone marrow inhibition or bonemarrow resection therapy for cancer patients.

On the other hand modified antibodies, especially antibodies withlowered molecular size, for example, single chain Fvs were developed toimprove permeability into tissues and tumors by lowering molecular sizeand to produce by a recombinant method. Recently the dimers of singlechain Fvs, especially bispecific-dimers have been used for crosslinkingcells. Typical examples of such dimers are hetero-dimers of single chainFvs recognizing antigens of cancer cells and antigens of host cells likeNK cells and neutrophils (Kipriyanov et al., Int. J. Cancer, 77,9763-9772, 1998). They were produced by construction technique of singlechain Fv as modified antibodies, which are more effective in treatingcancers by inducing intercellular crosslinking. It has been thought thatthe intercellular crosslinking is induced by antibodies and theirfragments (e.g. Fab fragment), bispecific modified antibodies and evendimers of single chain Fvs, which are monospecific.

As antibodies capable of transducing a signal by crosslinking a cellsurface molecule(s), there are known an antibody against EPO receptorinvolved in cell differentiation and proliferation (JP-A 2000-95800), anantibody against MuSK receptor (Xie et al., Nature Biotech. 15, 768-771,1997) and others. There are also known an agonist antibody to TPOreceptor, its fragments and single chain Fvs (WO99/17364). However therehave been no reports on single chain Fv dimers and modified antibodiessuch as single chain bivalent antibodies having agonist activity.

Noticing that single chain Fv monomers derived from monoclonalantibodies (antibody MABL-1 and antibody MABL-2 produced by theinventors) which induce apoptosis of IAP-containing cells do not induceapoptosis of cells and that dimers induce apoptosis, the inventorsdiscovered that dimers crosslink (dimerize) IAP receptor on cellsurface, thereby a signal is transduced into the cells and, as a result,apoptosis is induced. This suggests that monospecific single chain Fvdimers crosslink a cell surface molecule(s) (e.g. receptor) andtransduce a signal like a ligand, thereby serving as an agonist.

Focusing on the intercellular crosslinking, it was discovered that theabove-mentioned single chain Fv dimers do not cause hemagglutinationwhile the above-mentioned monoclonal antibodies do. The same result wasalso observed with single chain bivalent antibodies (single chainpolypeptides containing two H chain V regions and two L chain Vregions). This suggests that monoclonal antibodies may formintercellular crosslinking while modified antibodies like single chainFv dimers and single chain bivalent antibodies crosslink a cell surfacemolecule(s) but do not form intercellular crosslinking.

Based on those observations the inventors have newly discovered thatmodified antibodies such as single chain Fv dimers and single chainbivalent antibodies crosslink a cell surface molecule(s) orintracellular molecule(s) of the same cell, in addition to knownintercellular crosslinking, and are suitable as a ligand to themolecule(s) (especially as a ligand which mimics the action of naturalligand).

Discovering further that an antibody molecule (whole IgG) can bemodified into single chain Fv dimers, single chain bivalent antibodiesand the like which crosslink a cell surface molecule(s), therebyreducing side effects caused by intercellular crosslinking and providingnew medicines inducing only desired effect on the cell, the inventorscompleted the invention. The modified antibodies of the invention haveremarkably high activity compared with whole antibodies (IgG) having thesame V region as the modified antibodies. They have an improvedpermeability into tissues due to the lowered molecular size comparedwith antibody molecules and the lack of constant regions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the result of flow cytometry, illustrating that human IgGantibody does not bind to L1210 cells expressing human IAP (hIAP/L1210).

FIG. 2 shows the result of flow cytometry, illustrating that the chimeraMABL-1 antibody specifically binds to L1210 cells expressing human IAP(hIAP/L1210).

FIG. 3 shows the result of flow cytometry, illustrating that the chimeraMABL-2 antibody specifically binds to L1210 cells expressing human IAP(hIAP/L1210).

FIG. 4 schematically illustrates the process for producing the singlechain Fv according to the present invention.

FIG. 5 illustrates a structure of an expression plasmid which can beused to express a DNA encoding the single chain Fv of the invention inE. coli. The (Gly₄Ser)₃ linker is shown in SEQ ID NO: 118.

FIG. 6 illustrates a structure of an expression plasmid which is used toexpress a DNA encoding the single chain Fv of the invention in mammaliancells. The (Gly₄Ser)₃ linker is shown in SEQ ID NO: 118.

FIG. 7 shows the result of western blotting in Example 5.4.

FIG. 8 shows the result of flow cytometry, illustrating that an antibodyin the culture supernatant of pCHO1/COS7 cell as a control does not bindto pCOS1/L1210 cell as a control.

FIG. 9 shows the result of flow cytometry, illustrating that an antibodyin the culture supernatant of MABL2-scFv/COS7 cells does not bind topCOS1/L1210 cells as a control.

FIG. 10 shows the result of flow cytometry, illustrating that anantibody in the culture supernatant of pCOS1/COS7 cells as a controldoes not bind to hIAP/L1210 cells.

FIG. 11 shows the result of flow cytometry, illustrating that anantibody in the culture supernatant of MABL2-scFv/COS7 cellsspecifically binds to hIAP/L1210 cells.

FIG. 12 shows the result of the competitive ELISA in Example 5.6.

FIG. 13 shows the results of the apoptosis-inducing effect in Example5.7, illustrating that the antibody in the culture supernatant ofpCHO1/COS7 cells as a control does not induce the apoptosis ofpCOS1/L1210 cells as a control.

FIG. 14 shows the results of the apoptosis-inducing effect in Example5.7, illustrating that the antibody in the culture supernatant ofMABL2-scFv/COS7 cells does not induce apoptosis of pCOS1/L1210 cells asa control.

FIG. 15 shows the results of the apoptosis-inducing effect in Example5.7, illustrating that the antibody in the culture supernatant ofpCHO1/COS7 cells as a control does not induce apoptosis of hIAP/L1210cells.

FIG. 16 shows the results of the apoptosis-inducing effect in Example5.7, illustrating that the antibody in the culture supernatant ofMABL2-scFv/COS7 cells specifically induces apoptosis of hIAP/L1210cells.

FIG. 17 shows the results of the apoptosis-inducing effect in Example5.7, illustrating that the antibody in the culture supernatant ofpCHO1/COS7 cells as a control does not induce apoptosis of CCRF-CEMcells.

FIG. 18 shows the results of the apoptosis-inducing effect in Example5.7, illustrating that the antibody in the culture supernatant ofMABL2-scFv/COS7 cells specifically induces apoptosis of CCRF-CEM cells.

FIG. 19 shows the chromatogram obtained in the purification of thesingle chain Fv derived form the antibody MABL-2 produced by the CHOcells in Example 5.9, illustrating that fraction A and fraction B wereobtained as the major peaks when the fraction from Blue-sepharose columnwas purified with hydroxyapatite column.

FIG. 20 shows the results of purification by gel filtration of fractionA and fraction B obtained in Example 5.9-(2), illustrating that themajor peaks (AI and BI, respectively) were eluted from fraction A atapproximately 36 kD of the apparent molecular weight and from fraction Bat approximately 76 kD.

FIG. 21 is the analysis on SDS-PAGE of the fractions obtained in thepurification of the single chain Fv derived from the antibody MABL-2produced by the CHO cells in Example 5.9.

FIG. 22 shows the results of analysis of fractions AI and BI obtained bygel filtration in the purification of the single chain Fv derived fromthe antibody MABL-2 produced by the CHO cells.

FIG. 23 illustrates a structure of an expression plasmid which can beused to express a DNA encoding the single chain Fv of the invention inE. coli. The (Gly₄Ser)₃ linker is shown in SEQ ID NO: 118.

FIG. 24 shows the results of purification on the gel filtration columnof crude products of the single chain Fv polypeptide derived from theantibody MABL-2 produced by E. coli obtained in Example 5.12.

FIG. 25 shows the results of the apoptosis-inducing effect in Example5.13, illustrating that mouse IgG antibody as a control does not induceapoptosis of hIAP/L1210 cells.

FIG. 26 shows the results of the apoptosis-inducing effect in Example5.13, illustrating that the dimer of MABL2-scFv produced by the CHOcells remarkably induces apoptosis of hIAP/L1210 cells.

FIG. 27 shows the results of the apoptosis-inducing effect in Example5.13, illustrating that the dimer of MABL2-scFv produced by E. coliremarkably induces apoptosis of hIAP/L1210 cells.

FIG. 28 shows the results of the apoptosis-inducing effect in Example5.13, illustrating that apoptosis induction to hIAP/L1210 cells by theMABL2-scFv monomer produced by the CHO cells is the same level as thatof the control.

FIG. 29 shows the results of the apoptosis-inducing effect in Example5.13, illustrating that apoptosis induction to hIAP/L1210 cells of theMABL2-scFv monomer produced by E. coli is the same level as that ofcontrol.

FIG. 30 shows the results of the apoptosis-inducing effect in Example5.13, illustrating that mouse IgG antibody used as a control does notinduce apoptosis of hIAP/L1210 cells even when anti-FLAG antibody isadded.

FIG. 31 shows the results of the apoptosis-inducing effect in Example5.13, illustrating that MABL2scFv monomer produced by the CHO cellsremarkably induces apoptosis of hIAP/L1210 cells when anti-FLAG antibodyis added.

FIG. 32 shows the results of quantitative measurement of human IgG inthe serum of a human myeloma cell line KPMM2-transplanted mouse.

FIG. 33 shows the survival time of the mouse after the transplantationof tumor

FIG. 34 illustrates a structure of an expression plasmid which expressesa modified antibody [sc(Fv)₂]. The (G₄S)₃ linker is shown in SEQ ID NO:118 while the (G₄S)₃VDS linker is shown in SEQ ID NO: 145.

FIG. 35 illustrates a structure of a plasmid which expresses a scFv (HLtype).

FIG. 36 illustrates a structure of the HL-type nucleotide and amino acidsequences of peptide linkers (SEQ ID NOS 148-159, respectively, in orderof appearance).

FIG. 37 illustrates a structure of a plasmid which expresses a scFv (LHtype) wherein the V regions are linked in the manner of [L chain]-[Hchain] without a peptide linker.

FIG. 38 illustrates a structure of the LH-type nucleotide and amino acidsequences of peptide linkers (SEQ ID NOS 162-173, respectively, in orderof appearance).

FIG. 39 shows the results of the western blotting in Example 6.4.

FIGS. 40 a and 40b show the results of flow cytometry using the culturesupernatant of COST cells prepared in Example 6.3 (1).

FIG. 41 shows the results of the apoptosis-inducing effect in Example6.6.

FIG. 42 shows the results of the evaluation of antigen binding capacityin Example 6.10.

FIG. 43 shows the results of the in vitro apoptosis-inducing effect inExample 6.11.

FIG. 44 shows the results of the quantitative measurement of M proteinproduced by a human myeloma cell line KPMM2 in the serum of the humanmyeloma celltransplanted mouse.

FIG. 45 shows the survival time (days) of mice after the transplantationof tumor.

FIG. 46 shows the survival time (days) of mice after the transplantationof tumor.

FIG. 47 is a scheme showing the method for constructing DNA fragmentencoding the reconstructed 12B5 single chain Fv containing the linkersequence. The (Gly₄Ser)₃ linker is shown in SEQ ID NO: 118.

FIG. 48 shows the purification result of each 12B5 single chain Fv bygel filtration obtained in Example 7.5 (1).

FIG. 49 shows the analytical result of each fraction A and B b SDS-PAGEperformed in Example 7.5 (2).

FIG. 50 shows the analytical result of each fraction A and B bySuperdex200 column performed in Example 7.5 (2).

FIG. 51 shows the measurement result of the TPO-like agonist activity ofsc12B5 and antibody 12B5.

FIG. 52 shows the measurement result of TOP-like agonist activity ofsc12B5 monomer and dimer.

FIG. 53 shows the purification result of obtained sc12E10 single chainantibody by gel filtration chromatography using Superdex200HR column.

FIG. 54 shows the purification result of obtained db12E10 single chainantibody by gel filtration chromatography using Superdex200HR column.

FIG. 55 shows SDS-PAGE analysis of fractions A and B (sc12E10) andfractions C and D (db12E10) under the reductive or non-reductivecondition.

FIG. 56 shows the analytical result of fractions A and B by gelfiltration chromatography using Superdex200HR column.

FIG. 57 shows the analytical result of fractions C and D by gelfiltration chromatography using Superdex200HR column.

FIG. 58 is a graph showing the agonist activity of various 12E10antibody molecules on MPL, illustrating that single chain Fvs (sc12E10,db12E10).

FIG. 59 is a graph showing the agonist activity of monomer and dimer ofsc12E10 and dimer and trimer of db12E10 on MPL.

DISCLOSURE OF INVENTION

An object of this invention is to provide low molecular-sized agonisticmodified antibodies which contain two or more H chain V regions and twoor more L chain V regions of a monoclonal antibody and have TPO agonistaction by crosslinking TPO receptor.

Therefore, this invention relates the modified antibodies which containtwo or more H chain V regions and two or more L chain V regions,preferably 2 to 6 each, especially preferably 2 to 4 each, mostpreferably two each, and show TPO agonist activity by crosslinking TPOreceptor.

The “modified antibodies” in the specification mean any substances whichcontain two or more H chain V regions and two or more L chain V regions,wherein said V regions are combined directly or via linker throughcovalent bond or non-covalent bond. For example, polypeptides andcompounds produced by combining each V region of antibody through apeptide linker or a chemical crosslinking agent and the like. Two ormore H chain V regions and two or more L chain V regions used in theinvention can be derived from the same antibody or from differentantibodies.

Modified antibodies of the invention can be any things as long as theyspecifically recognize and crosslink TPO receptor and thereby cantransduce a signal into cells. They include modified antibodies producedby further modifying a part of the amino acid sequence of V region ofthe modified antibodies.

Preferable examples of the modified antibodies of the invention aremultimers such as dimers, trimers or tetramers of single chain Fvcontaining an H chain V region and an L chain V region, or single chainpolypeptides containing two or more H chain V regions and two or more Lchain V regions. When the modified antibodies of the invention aremultimers of single chain Fv such as dimers, trimers, tetramers and thelike containing an H chain V region and an L chain V region, it ispreferable that the H chain V region and L chain V region existing inthe same chain are not associated to form an antigen-binding site.

More preferable examples are dimers of the single chain Fv whichcontains an H chain V region and an L chain V region, or a single chainpolypeptide containing two H chain V regions and two L chain V regions.The H chain V region and L chain V region are connected preferablythrough a linker in the modified antibodies.

The above-mentioned single chain Fv multimer includes a multimer bynon-covalent bond, a multimer by a covalent bond through a crosslinkingradical and a multimer through a crosslinking reagent (an antibody, anantibody fragment, or bivalent modified antibody). Conventionalcrosslinking radicals used for crosslinking peptides can be used as thecrosslinking radicals to form the multimers. Examples are disulfidecrosslinking by cysteine residue, other crosslinking radicals such asC₄-C₁₀ alkylene (e.g. tetramethylene, pentamethylene, hexamethylene,heptamethylene and octamethylene, etc.) or C₄-C₁₀ alkenylene(cis/trans-3-butenylene, cis/trans-2-pentenylene,cis/trans-3-pentenylene, cis/trans-3-hexenylene, etc.).

Moreover, the crosslinking reagent which can combine with a single chainFv is, for example, an amino acid sequence which can optionally beintroduced into Fv, for example, an antibody against FLAG sequence andthe like or a fragment thereof, or a modified antibody originated fromthe antibody, for example, single chain Fv.

“TPO agonist action” in the specification means a biological actionoccurring in the cell(s) into which a signal is transduced bycrosslinking TPO receptor, for example, proliferation, differentiationor growth stimulation of megakaryocytes, or platelet production.

ED50 of the TPO agonist action in the invention is determined by knownmethods for measuring agonist action. Examples for measurement are cellproliferation assay using TPO sensitive cell lines such as BaF/mpl orUT7/TPO, measurement of phosphorylation of MPL protein, megakaryocytecolony assay by differentiation from bone marrow cells, in vivo mouseplatelet recovery synthesis assay, measurement of expression inductionof platelet antigen GPIIbIIIa (anti GPIIbIIIa) using human leukemiamegakaryoblastic cell line (CMK) or measurement of polyploidy inductionof megakaryoblastic cell line (DAMI). ED50 is a dose needed forachieving 50% reaction of the maximum activity set as 100% in thedose-reaction curve.

Preferable modified antibodies of the invention have TPO agonist action(ED50) equivalent to or better than that of an antibody having the sameantigen-binding region as the modified antibody, namely the wholeantibody (hereinafter “parent antibody”) like IgG having the same pairof H chain V region and L chain V region as the pair of H chain V regionand L chain V region forming antigen-biding region of the modifiedantibody. More preferable are those having TPO agonist action (ED50)more than two times higher than that of parent antibody, furtherpreferably more than 5 times, most preferably more than 10 times. Theinvention includes modified antibodies with TPO agonist actioncontaining H chain V region and L chain V region forming the sameantigen-binding region as the parent antibody which binds to TPOreceptor but has no TPO agonist action to the molecule.

The compounds containing two or more H chain V regions and two or more Lchain V regions of the invention can be any compounds which contain twoor more H chain V regions and two or more L chain V regions of antibodyand show TPO agonist action (ED50) equivalent to or better than that ofthrombopoietin (TPO). Preferable are those having TPO agonist action(ED50) more than two times higher than that of TPO, more preferably morethan 5 times, most preferably more than 10 times.

The “compounds” mentioned here include not only modified antibodies ofthe invention but also any compounds containing two or more, preferablyfrom 2 to 6, more preferably from 2 to 4, most preferably 2antigen-binding regions such as whole antibodies or F(ab′)₂.

Preferable modified antibodies or compounds of the invention containingtwo or more H chain V regions and two or more L chain V regions ofantibody have an intercellular adhesion action (ED50) not more than 1/10compared with the parent antibody, more preferably have no substantialintercellular adhesion action.

ED50 of the intercellular adhesion action mentioned in the above isdetermined by known methods for measuring intercellular adhesion action,for example, by the measurement of agglomeration of cells expressing TPOreceptor.

The invention relates to DNAs which code for the modified antibodies.

The invention relates to animal cells or microorganisms which producethe modified antibodies.

The invention relates to use of the modified antibody as TPO agonist.

The invention relates to a method of transducing a signal into cells bycrosslinking TPO receptor using the modified antibody and therebyinducing TPO agonist action such as proliferation,differentiation-induction or growth stimulation of megakaryocytes,platelet production, phosphorylation of TPO receptor protein and thelike.

The invention relates to a medicine for treating thrombocytopenia etc.containing the modified antibody as active component.

The invention relates to use of the modified antibody as a medicine.

The invention relates to a method of screening or measuring the modifiedantibody, which contains two or more H chain V regions and two or more Lchain V regions of antibody and shows TPO agonist action by crosslinkingTPO receptor, that comprises 1) to prepare a modified antibodycontaining two or more H chain V regions and two or more L chain Vregions of antibody and binding specifically to TPO receptor, 2) tocontact the modified antibody with cells expressing TPO receptor and 3)to measure TPO agonist action which occurs in the cells by crosslinkingTPO receptor. The method of measurement is useful for the qualitycontrol in producing the modified antibodies of the invention as amedicine and other purposes.

The modified antibodies can be mono-specific modified antibodies ormulti-specific modified antibodies like bi-specific modified antibodies.Preferable are mono-specific modified antibodies.

The present invention also relates to modified antibodies whose H chainV region and/or L chain V region is H chain V region derived from humanantibody and/or L chain V region derived from human antibody. The Hchain V region and/or L chain V region derived from human antibody canbe obtained by screening human monoclonal antibody library as describedin WO99/10494. The H chain V region and L chain V region derived fromhuman monoclonal antibodies produced by transgenic mouse and the likeare also included.

The present invention further relates to modified antibodies whose Hchain V regions and/or L chain V regions are humanized H chain V regionsand/or humanized L chain V regions. Specifically, the humanized modifiedantibodies consist of the humanized L chain V region which comprisesframework regions (FR) derived from an L chain V region of humanmonoclonal antibody and complementarity determining regions (hereinafter“CDR”) derived from an L chain V region of non-human mammalian (e.g.mouse, rat, bovine, sheep, ape) monoclonal antibody and/or the humanizedH chain V region which comprises FR derived from an H chain V region ofhuman monoclonal antibody and CDR derived from an H chain V region ofnon-human mammalian (e.g. mouse, rat, bovine, sheep, ape) monoclonalantibody. In this case, the amino acid sequences of CDR and FR may bepartially altered, e.g. deleted, replaced or added.

H chain V regions and/or L chain V regions of the modified antibodies ofthe invention can be H chain V regions and/or L chain V regions derivedfrom monoclonal antibodies of animals other than human (such as mouse,rat, bovine, sheep, ape, chicken and the like). In this case, the aminoacid sequences of CDR and FR may be partially altered, e.g. deleted,replaced or added.

The invention also relates to DNAs encoding the various modifiedantibodies as mentioned above and genetic engineering techniques forproducing recombinant vectors comprising the DNAs.

The invention also relates to host cells transformed with therecombinant vectors. Examples of host cells are animal cells such ashuman cells, mouse cells or the like and microorganisms such as E. coli,Bacillus subtilis, yeast or the like.

The invention relates to a process for producing the modifiedantibodies, which comprises culturing the above-mentioned hosts andextracting the modified antibodies from the culture thereof.

The present invention further relates to a process for producing a dimerof the single chain Fv which comprises culturing host animal cellsproducing the single chain Fv in a serum-free medium to secrete thesingle chain Fv into the medium and isolating the dimer of the singlechain Fv formed in the medium.

The present invention also relates to the use of the modified antibodiesas TPO agonist. That is, it relates to a signal-transduction agonistwhich comprises as an active ingredient the modified antibody obtainedas mentioned in the above.

Therefore, the pharmaceutical preparations containing TPO agonistmodified antibodies of the invention as an active ingredient are usefulas preventives and/or remedies for platelet-reduction-related blooddiseases, thrombocytopenia caused by chemotherapy of cancers orleukemia, and the like.

The modified antibodies of the present invention comprise two or more Hchain V regions and two or more L chain V regions derived fromantibodies. The structure of the modified antibodies may be a dimer ofsingle chain Fv comprising one H chain V region and one L chain V regionor a polypeptide comprising two H chain V regions and two L chain Vregions. In the modified antibodies of the invention, the V regions of Hchain and L chain are preferably linked through a peptide linker whichconsists of one or more amino acids. The resulting modified antibodiescontain variable regions of antibodies and bind to the antigen with thesame specificity as that of the original monoclonal antibodies.

H Chain V Region

In the present invention, the H chain V region derived from an antibodyrecognizes TPO receptor and oligomerizes, for example, dimerizes throughcrosslinking said molecule, and thereby transduces a signal into thecells. The H chain V region of the invention includes H chain V regionsderived from a mammal (e.g. human, mouse, rat, bovine, sheep, ape etc.)and H chain V regions having partially modified amino acid sequences ofthe H chain V regions. More preferable is a humanized H chain V regioncontaining FR of H chain V region of a human monoclonal antibody and CDRof H chain V region of a mouse monoclonal antibody. Also preferable isan H chain V region having an amino acid sequence derived from a human,which can be produced by recombination technique. The H chain V regionof the invention may be a fragment of aforementioned H chain V region,which fragment preserves the antigen binding capacity.

L Chain V Region

In the present invention, the L chain V region recognizes TPO receptorand oligomerizes, for example, dimerizes through crosslinking saidmolecule, and thereby transduces a signal into the cells. The L chain Vregion of the invention includes L chain V regions derived from a mammal(e.g. human, mouse, rat, bovine, sheep, ape etc.) and L chain V regionshaving partially modified amino acid sequences of the L chain V regions.More preferable is a humanized L chain V region containing FR of L chainV region of human monoclonal antibody and CDR of L chain V region ofmouse monoclonal antibodies. Also preferable is an L chain V regionhaving an amino acid sequence derived from a human, which can beproduced by recombination technique. The L chain V regions of theinvention may be fragments of L chain V region, which fragments preservethe antigen binding capacity.

Complementarity Determining Region (CDR)

Each V region of L chain and H chain forms an antigen-binding site. Thevariable region of the L and H chains is composed of comparativelyconserved four common framework regions linked to three hypervariableregions or complementarity determining regions (CDR) (Kabat, E. A. etal., “Sequences of Protein of Immunological Interest”, US Dept. Healthand Human Services, 1983).

Major portions in the four framework regions (FRs) form β-sheetstructures and thus three CDRs form a loop. CDRs may form a part of theβ-sheet structure in certain cases. The three CDRs are held stericallyclose position to each other by FR, which contributes to the formationof the antigen-binding site together with three CDRs.

These CDRs can be identified by comparing the amino acid sequence of Vregion of the obtained antibody with known amino acid sequences of Vregions of known antibodies according to the empirical rule in Kabat, E.A. et al., “Sequences of Protein of Immunological Interest”.

Single Chain Fv

A single chain Fv is a polypeptide monomer comprising an H chain Vregion and an L chain V region linked each other which are derived fromantibodies. The resulting single chain Fvs contain variable regions ofthe original antibodies and preserve the complementarity determiningregion thereof, and therefore the single chain Fvs bind to the antigenby the same specificity as that of the original antibodies (JP-Appl.11-63557). A part of the variable region and/or CDR of the single chainFv of the invention or a part of the amino acid sequence thereof may bepartially altered, for example, deleted, replaced or added. The H chainV region and L chain V region composing the single chain Fv of theinvention are mentioned before and may be linked directly or through alinker, preferably a peptide linker. The constitution of the singlechain Fv may be [H chain V region]-[L chain V region] or [L chain Vregion]-[H chain V region]. In the present invention, it is possible tomake the single chain Fv to form a dimer, a trimer or a tetramer, fromwhich the modified antibody of the invention can be formed.

Single Chain Modified Antibody

The single chain modified antibodies of the present invention comprisingtwo or more H chain V regions and two or more L chain V regions,preferably each two to four, especially preferable each two, comprisetwo or more H chain V regions and L chain V regions as mentioned above.Each region of the peptide should be arranged such that the modifiedsingle chain antibody forms a specific steric structure, concretelymimicking a steric structure formed by the dimer of single chain Fv. Forinstance, the V regions are arranged in the order of the followingmanner:

-   [H chain V region]-[L chain V region]-[H chain V region]-[L chain V    region]; or-   [L chain V region]-[H chain V region]-[L chain V region]-[H chain V    region],    wherein these regions are connected through a peptide linker,    respectively.    Linker

In this invention, the linkers for the conMection between the H chain Vregion and the L chain V region may be any peptide linker which can beintroduced by the genetic engineering procedure or any linker chemicallysynthesized. For instance, linkers disclosed in literatures, e.g.Protein Engineering, 913), 299-305, 1996 may be used in the invention.These linkers can be the same or different in the same molecule. Ifpeptide linkers are required, the following are cited as examplelinkers:

Ser Gly-Ser Gly-Gly-Ser Ser-Gly-Gly Gly-Gly-Gly-Ser (SEQ ID NO: 174)Ser-Gly-Gly-Gly (SEQ ID NO: 175) Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 176)Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 177) Gly-Gly-Gly-Gly-Gly-Ser(SEQ ID NO: 178) Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 179)Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 180) Ser-Gly-Gly-Gly-Gly-Gly-Gly(SEQ ID NO: 181) (Gly-Gly-Gly-Gly-Ser)_(n )and (SEQ ID NO: 182)(Ser-Gly-Gly-Gly-Gly)_(n) (SEQ ID NO: 183)wherein n is an integer not less than one. Preferable length of thelinker peptide varies dependent upon the receptor to be the antigen, inthe case of single chain Fvs, the range of 1 to 20 amino acids isnormally preferable. In the case of single chain modified antibodiescomprising two or more H chain V regions and two or more L chain Vregions, the peptide linkers conMecting those forming the same antigenbinding site comprising [H chain V region]-[L chain V region] (or [Lchain V region]-[H chain V region]) have lengths of 1-30 amino acids,preferably 1-20 amino acids, more preferably 3-18 amino acids. Thepeptide linkers conMecting those not forming the same antigen bidingsite comprising [H chain V region]-[L chain V region] or ([L chain Vregion]-[H chain V region]) have lengths of 1-40 amino acids, preferably3-30 amino acids, more preferably 5-20 amino acids. The method forintroducing those linkers will be described in the explanation for DNAconstruction coding for modified antibodies of the invention.

The chemically synthesized linkers, i.e. the chemical crosslinkingagents, according to the invention can be any linkers conventionallyemployed for the linkage of peptides. Examples of the linkers mayinclude N-hydroxy succinimide (NHS), disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl)suberate (BS³), dithiobis(succinimidyl propionate)(DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethyleneglycolbis(succinimidyl succinate) (EGS), ethyleneglycolbis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyltartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),bis[2-(succinimido oxycarbonyloxy)ethyl]sulfone (BSOCOES),bis[2-(sulfosuccinimido oxycarbonyloxy) ethyl]sulfone (sulfo-BSOCOES) orthe like. These are commercially available. It is preferable for thechemically synthesized linkers to have the length equivalent to that ofpeptide linkers.

To form a dimer of the single chain Fv it is preferable to select alinker suitable to dimerize in the solution such as culture medium morethan 20%, preferably more than 50%, more preferably more than 80%, mostpreferably more than 90% of the single chain Fv produced in the hostcells. Specifically, preferable is a linker composed of 2 to 12 aminoacids, preferably 3 to 10 amino acids or other linkers correspondingthereto.

Preparation of Modified Antibodies

The modified antibodies can be produced by connecting, through theaforementioned linker, an H chain V region and an L chain V regionderived from known or novel antibodies specifically binding to TPOreceptor. As examples of the single chain Fvs are cited those having Hchain V region and L chain V region of antibody 12B5 and antibody 12E10described in WO99/10494. As examples of the modified antibodies of theinvention having two or more H chain V regions and two or more L chain Vregions are cited sc12B5 dimer (linker: 15 amino acids), sc12E10 dimer(linker: 15 amino acids), db12B5 dimer (linker: 5 amino acids), db12E10dimer (linker: 5 amino acids), sc12B5sc(FV) 2 and sc12E10sc(FV)₂ whichcontain H chain V regions and L chain V regions derived from theabove-mentioned monoclonal antibodies.

For the preparation of the modified antibodies, a signal peptide may beattached to its N-terminal if the polypeptide is desired to be asecretory peptide. A well-known amino acid sequence useful for thepurification of polypeptide such as the FLAG sequence may be attachedfor the efficient purification of the polypeptide. In this case a dimercan be formed by using an ANTI-FLAG anti FLAG antibody.

For the preparation of the modified antibody of the invention, it isnecessary to obtain a DNA, i.e. a DNA encoding the single chain Fv or aDNA encoding reconstructed single chain polypeptide. These DNAs,especially for sc12B5, db12B5, sc12E10 and/or db12E10 are obtainablefrom the DNAs encoding the H chain V regions and the L chain V regionsderived from said Fvs. They are also obtainable by polymerase chainreaction (PCR) method using those DNAs as a template and amplifying thepart of DNA contained therein encoding desired amino acid sequence withthe aid of a pair of primers corresponding to both ends thereof.

In the case where each V region having partially modified amino acidsequence is desired, the V regions in which one or some amino acids aremodified, i.e. deleted, replaced or added can be obtained by a procedureknown in the art using PCR. A part of the amino acid sequence in the Vregion is preferably modified by the PCR known in the art in order toprepare the modified antibody which is sufficiently active against thespecific antigen.

For the determination of primers for the PCR amplification, the types ofH chain and L chain, if a monoclonal antibody is used as a startingmaterial, are determined by a typing method known in the technicalfield.

For the amplification of the L chain V regions of antibody 12B5 andantibody 12E10 by PCR, 5′-end and 3′-end oligonucleotide primers aredecided as aforementioned. In the same manner, 5′-end and 3′-endoligonucleotide primers are decided for the amplification of the H chainV regions of antibody 12B5 and antibody 12E10.

In embodiments of the invention, the 5′-end primers which contain asequence “GANTC” providing the restriction enzyme Hinf I recognitionsite at the neighborhood of 5′-terminal thereof are used and the 3′-endprimers which contain a nucleotide sequence “CCCGGG” providing the XmaIrecognition site at the neighborhood of 5′-terminal thereof are used.Other restriction enzyme recognition site may be used instead of thesesites as long as they are used for subcloning a desired DNA fragmentinto a cloning vector.

Specifically designed PCR primers are employed to provide suitablenucleotide sequences at 5′-end and 3′-end of the cDNAs encoding the Vregions of the antibodies 12B5 and 12E10 so that the cDNAs are readilyinserted into an expression vector and appropriately function in theexpression vector (e.g. this invention devises to increase transcriptionefficiency by inserting Kozak sequence). The V regions of the antibodies12B5 and 12E10 obtained by amplifying by PCR using these primers areinserted into HEF expression vector containing the desired human Cregion (see WO92/19759). The cloned DNAs can be sequenced by using anyconventional process, for example, by the automatic DNA sequencer(Applied Biosystems).

A linker such as a peptide linker can be introduced into the modifiedantibody of the invention in the following manner. Primers which havepartially complementary sequence with the primers for the H chain Vregions and the L chain V regions as described above and which code forthe N-terminal or the C-terminal of the linker are designed. Then, thePCR procedure can be carried out using these primers to prepare a DNAencoding the peptide linker having desired amino acid sequence andlength. The DNAs encoding the H chain V region and the L chain V regioncan be connected through the resulting DNA to produce the DNA encodingthe modified antibody of the invention which has the desired peptidelinker. Once the DNA encoding one of the modified antibodies isprepared, the DNAs encoding the modified antibodies with or without thedesired peptide linker can readily be produced by designing variousprimers for the linker and then carrying out the PCR using the primersand the aforementioned DNA as a template.

Each V region of the modified antibody of the present invention can behumanized by using conventional techniques (e.g. Sato, K. et al., CancerRes., 53, 1-6 (1993)). Once a DNA encoding each of humanized Fvs isprepared, a humanized single chain Fv, a fragment of the humanizedsingle chain Fv, a humanized monoclonal antibody and a fragment of thehumanized monoclonal antibody can readily be produced according toconventional methods. Preferably, amino acid sequences of the V regionsthereof may be partially modified, if necessary.

Furthermore, a DNA derived from other mammalian origin, for example aDNA encoding each of V regions of human antibody, can be produced in thesame manner as used to produce DNA encoding the H chain V region and theL chain V region derived from mouse by conventional methods as mentionedin the above. The resulting DNA can be used to prepare an H chain Vregion and an L chain V region of other mammal, especially derived fromhuman antibody, a single chain Fv derived from human and a fragmentthereof, and a monoclonal antibody of human origin and a fragmentthereof.

When the modified antibodies of the invention is bi-specific modifiedantibodies, they can be produced by known methods (for example, themethod described in WO9413804).

As mentioned above, when the aimed DNAs encoding the V regions of themodified antibodies and the V regions of the humanized modifiedantibodies are prepared, the expression vectors containing them andhosts transformed with the vectors can be obtained according toconventional methods. Further, the hosts can be cultured according to aconventional method to produce the reconstructed single chain Fv, thereconstructed humanized single chain Fv, the humanized monoclonalantibodies and fragments thereof. They can be isolated from cells or amedium and can be purified into a homogeneous mass. For this purpose anyisolation and purification methods conventionally used for proteins,e.g. chromatography, ultra-filtration, salting-out and dialysis, may beemployed in combination, if necessary, without limitation thereto.

When the reconstructed single chain Fv of the present invention isproduced by culturing an animal cell such as COS7 cells or CHO cells,preferably CHO cells, in a serum-free medium, the dimer of said singlechain Fv formed in the medium can be stably recovered and purified in ahigh yield. Thus purified dimer can be stably preserved for a longperiod. The serum-free medium employed in the invention may be anymedium conventionally used for the production of a recombinant proteinwithout limit thereto.

For the production of the modified antibodies of the present invention,any expression systems can be employed, for example, eukaryotic cellssuch as animal cells, e.g., established mammalian cell lines,filamentous fungi and yeast, and prokaryotic cells such as bacterialcells e.g., E. coli. Preferably, the modified antibodies of theinvention are expressed in mammalian cells, for example COS7 cells orCHO cells.

In these cases, conventional promoters useful for the expression inmammalian cells can be used. Preferably, human cytomegalovirus (HCMV)immediate early promoter is used. Expression vectors containing the HCMVpromoter include HCMV-VH-HCγ 1, HCMV-VL-HCK and the like which arederived from pSV2neo (WO92/19759).

Additionally, other promoters for gene expression in mammal cell whichmay be used in the invention include virus promoters derived formretrovirus, polyoma virus, adenovirus and simian virus 40 (SV40) andpromoters derived from mammal such as human polypeptide-chain elongationfactor-1α (HEF-1α). SV40 promoter can easily be used according to themethod of Mulligan, R. C., et al. (Nature 277, 108-114 (1979)) andHEF-1α promoter can also be used according to the methods of Mizushima,S. et al. (Nucleic Acids Research, 18, 5322 (1990)).

Replication origin (ori) which can be used in the invention includes oriderived from SV40, polyoma virus, adenovirus, bovine papilloma virus(BPV) and the like. An expression vector may contain, as a selectionmarker, phosphotransferase APH (3′) II or I (neo) gene, thymidine kinase(TK) gene, E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt)gene or dihydrofolate reductase (DHFR) gene.

The antigen-binding activity of the modified antibody prepared in theabove can be evaluated by a conventional method such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) or surfaceplasmon resonance. It can also be evaluated using the binding-inhibitoryability of original antibody as an index, for example in terms of theabsence or presence of concentration-dependent inhibition of the bindingof said monoclonal antibody to the antigen.

More in detail, animal cells transformed with an expression vectorcontaining a DNA encoding the modified antibody of the invention, e.g.,COS7 cells or CHO cells, are cultured. The cultured cells and/or thesupernatant of the medium or the modified antibody purified from themare used to determine the binding to antigen. As a control is used asupernatant of the culture medium in which cells transformed only withthe expression vector were cultured. In the case of an antigen, forexample, the antibody 12B5 and the antibody 12E10, a test sample of themodified antibody of the invention or a supernatant of the control isadded to Ba/F3 cells expressing human MPL and then an assay such as theflow cytometry is carried out to evaluate the antigen-binding activity.

In vitro evaluation of the signal transduction effect (for example,proliferation, differentiation-induction or growth stimulation ofmegakaryocyte, platelet production, or phosphorylation of TPO receptorprotein) is performed in the following manner. A test sample of theabove-mentioned modified antibody is added to the cells which areexpressing the antibody or cells into which the gene for the antibodyhas been introduced, and is evaluated by the change caused by the signaltransduction (for example, human MPL antigen-specific proliferation,measurement of protein phosphorylation, or expression ofplatelet-specific antigen) using conventional methods.

In vivo evaluation is carried out by administering a monoclonal antibodyrecognizing MPL, a modified antibody of the invention and PBS as controlto mice, and evaluating the strength of the activity by the change ofthe amount of platelet in mouse serum.

As mentioned above the modified antibodies of the invention can beobtained by preparing modified antibodies which contain two or more Hchain V regions and two or more L chain V regions and specifically bindto TPO receptor and screening the modified antibodies by in vivo or invitro evaluation as mentioned in the above.

The modified antibodies of the invention, which comprises two or more Hchain V regions and two or more L chain V regions, preferably each twoto four, more preferably each two, may be a dimer of the single chain Fvcomprising one H chain V region and one L chain V region, or a singlechain polypeptide in which two or more H chain V regions and two or moreL chain V regions are connected. It is considered that owing to suchconstruction the peptide mimics three dimensional structure of TPO andtherefore retains an excellent antigen-binding property and TPO agonistactivity.

The modified antibodies of the invention have a remarkably loweredmolecular size compared with parent antibody molecule (e.g. IgG), and,therefore, have a superior permeability into tissues and tumors and ahigher activity than parent monoclonal antibody molecule. Therefore, themodified antibodies of the invention can efficiently transduce TPOsignal into cells. The pharmaceutical preparations containing them areuseful for treating platelet-reduction-related blood diseases andthrombocytopenia caused by chemotherapy for cancers or leukemia. It isfurther expected that the antibody of the invention can be used as acontrast agent by RI-labeling. The effect can be enhanced by attachingto a RI-compound or a toxin.

BEST MODE FOR WORKING THE INVENTION

The present invention will concretely be illustrated in reference to thefollowing examples, which in no way limit the scope of the invention.

For illustrating the production process of the modified antibodies ofthe invention, examples of producing single chain Fvs are shown below.Mouse antibodies against human IAP, MABL-1 and MABL-2 were used in theexamples of producing the modified antibodies. Hybridomas MABL-1 andMABL-2 producing them respectively were internationally deposited asFERM BP-6100 and FERM BP-6101 with the National Institute of Bioscienceand Human Technology, Agency of Industrial Science and Technology,Minister of International Trade and Industry (1-3 Higasi 1-chome,Tsukuba-shi, Ibaraki-ken, Japan), an authorized depository formicroorganisms, on Sep. 11, 1997.

EXAMPLES Example 1 Cloning of DNAs Encoding V Region of Mouse MonoclonalAntibodies to Human IAP

DNAs encoding variable regions of the mouse monoclonal antibodies tohuman IAP, MABL-1 and MABL-2, were cloned as follows.

1.1 Preparation of Messenger RNA (mRNA)

mRNAs of the hybridomas MABL-1 and MABL-2 were obtained by using mRNAPurification Kit (Pharmacia Biotech).

1.2 Synthesis of Double-Stranded cDNA

Double-stranded cDNA was synthesized from about 1 μg of the mRNA usingMarathon cDNA Amplification Kit (CLONTECH) and an adapter was linkedthereto.

1.3 PCR Amplification of Genes Encoding Variable Regions of an Antibodyby

PCR was carried out using Thermal Cycler (PERKIN ELMER).

(1) Amplification of a Gene Coding for L Chain V Region of MABL-1

Primers used for the PCR method are Adapter Primer-1 (CLONTECH) shown inSEQ ID No. 1, which hybridizes to a partial sequence of the adapter, andMKC (Mouse Kappa Constant) primer (Bio/Technology, 9, 88-89, 1991) shownin SEQ ID No. 2, which hybridizes to the mouse kappa type L chain Vregion.

50 μl of the PCR solution contains 5 μl of 10×PCR Buffer II, 2 mM MgCl₂,0.16 mM dNTPs (DATP, dGTP, dCTP and dTTP), 2.5 units of a DNApolymerase, AMPLITAQ GOLD (PERKIN ELMER), 0.2 μM of the adapter primerof SEQ ID No. 1, 0.2 μM of the MKC primer of SEQ ID No. 2 and 0.1 μg ofthe double-stranded cDNA derived from MABL-1. The solution was preheatedat 94° C. of the initial temperature for 9 minutes and then heated at94° C. for 1 minute, at 60° C. for 1 minute and at 72° C. for 1 minute20 seconds in order. This temperature cycle was repeated 35 times andthen the reaction mixture was further heated at 72° C. for 10 minutes.

(2) Amplification of cDNA Encoding H Chain V Region of MABL-1

The Adapter Primer-1 shown in SEQ ID No. 1 and MHC-γ1 (Mouse HeavyConstant) primer (Bio/Technology, 9, 8889, 1991) shown in SEQ ID No. 3were used as primers for PCR.

The amplification of cDNA was performed according to the method of theamplification of the L chain V region gene, which was described inExample 1.3-(1), except for using 0.2 μM of the MHC-γ1 primer instead of0.2 μM of the MKC primer.

(3) Amplification of cDNA Encoding L Chain V Region of MABL2

The Adapter Primer-1 of SEQ ID No. 1 and the MKC primer of SEQ ID No. 2were used as primers for PCR.

The amplification of cDNA was carried out according to the method of theamplification of the L chain V region gene of MABL-1 which was describedin Example 1.3-(1), except for using 0.1 μg of the double-stranded cDNAderived from MABL-2 instead of 0.1 μg of the double-stranded cDNA fromMABL-1.

(4) Amplification of cDNA Encoding H Chain V Region of MABL2

The Adapter Primer-1 of SEQ ID No. 1 and MHC-γ2a primer (Bio/Technology,9, 88-89, 1991) shown in SEQ ID No. 4 were used as primers for PCR.

The amplification of cDNA was performed according to the method of theamplification of the L chain V region gene, which was described inExample 1.3-(3), except for using 0.2 μM of the MHC-γ2a primer insteadof 0.2 μM of the MKC primer.

1.4 Purification of PCR Products

The DNA fragment amplified by PCR as described above was purified usingthe QIAquick PCR Purification Kit (QIAGEN) and dissolved in 10 mMTris-HCl (pH 8.0) containing 1 mM EDTA.

1.5 Ligation and Transformation

About 140 ng of the DNA fragment comprising the gene encoding the mousekappa type L chain V region derived from MABL-1 as prepared above wasligated with 50 ng of pGEM-T Easy vector (Promega) in the reactionbuffer comprising 30 mM Tris-HCl (pH 7.8), 10 mM MgCl₂, 10 mMdithiothreitol, 1 mM ATP and 3 units of T4 DNA Ligase (Promega) at 15°C. for 3 hours.

Then, 1 μl of the reaction mixture was added to 50 μl of E. coli DH5αcompetent cells (Toyobo Inc.) and the cells were stored on ice for 30minutes, incubated at 42° C. for 1 minute and stored on ice for 2minutes again. 100 μl of SOC medium (GIBCO BRL) was added. The cells ofE. coli were plated on LB (Molecular Cloning: A Laboratory Manual,Sambrook et al., Cold Spring Harbor Laboratory Press, 1989) agar mediumcontaining 100 μg/ml of ampicillin (SIGMA) and cultured at 37° C.overnight to obtain the transformant of E. coli.

The transformant was cultured in 3 ml of LB medium containing 50 μg/mlof ampicillin at 37° C. overnight and the plasmid DNA was prepared fromthe culture using the QIAprep Spin Miniprep Kit (QIAGEN).

The resulting plasmid comprising the gene encoding the mouse kappa typeL chain V region derived from the hybridoma MABL-1 was designated aspGEM-M1L.

According to the same manner as described above, a plasmid comprisingthe gene encoding the mouse H chain V region derived from the hybridomaMABL-1 was prepared from the purified DNA fragment and designated aspGEM-M1H.

A plasmid comprising the gene encoding the mouse kappa type L chain Vregion derived from the hybridoma MABL2 was prepared from the purifiedDNA fragment and designated as pGEM-M2L.

A plasmid comprising the gene encoding the mouse H chain V regionderived from the hybridoma MABL-2 was prepared from the purified DNAfragment and designated as pGEM-M2H.

Example 2 DNA Sequencing

The nucleotide sequence of the cDNA encoding region in theaforementioned plasmids was determined using Auto DNA Sequencer (AppliedBiosystem) and ABI PRISM Dye Terminator Cycle Sequencing Ready ReactionKit (Applied Biosystem) according to the manufacturer's protocol.

The nucleotide sequence of the gene encoding the L chain V region fromthe mouse antibody MABL-1, which is included in the plasmid pGEM-M1L, isshown in SEQ ID No. 5.

The nucleotide sequence of the gene encoding the H chain V region fromthe mouse antibody MABL-1, which is included in the plasmid pGEM-M1H, isshown in SEQ ID No. 6.

The nucleotide sequence of the gene encoding the L chain V region fromthe mouse antibody MABL-2, which is included in the plasmid pGEM-M2L, isshown in SEQ ID No. 7.

The nucleotide sequence of the gene encoding the H chain V region fromthe mouse antibody MABL-2, which is included in the plasmid pGEM-M2H, isshown in SEQ ID No. 8.

Example 3 Determination of CDR

The V regions of L chain and H chain generally have a similarity intheir structures and each four framework regions therein are linked bythree hypervariable regions, i.e., complementarity determining regions(CDR). An amino acid sequence of the framework is relatively wellconserved, while an amino acid sequence of CDR has extremely highvariation (Kabat, E. A., et al., “Sequences of Proteins of ImmunologicalInterest”, US Dept. Health and Human Services, 1983).

On the basis of these facts, the amino acid sequences of the variableregions from the mouse monoclonal antibodies to human IAP were appliedto the database of amino acid sequences of the antibodies made by Kabatet al. to investigate the homology. The CDR regions were determinedbased on the homology as shown in Table 1.

TABLE 1 Plasmid SEQ ID No. CDR(1) CDR(2) CDR(3) pGEM-M1L 5 43-58 74-80113-121 pGEM-M1H 6 50-54 69-85 118-125 pGEM-M2L 7 43-58 74-80 113-121pGEM-M2H 8 50-54 69-85 118-125

Example 4 Identification of Cloned cDNA Expression

(Preparation of Chimera MABL-1 antibody and Chimera MABL-2 antibody.)

4.1 Preparation of Vectors Expressing Chimera MABL-1 Antibody

cDNA clones, pGEM-M1L and pGEM-M1H, encoding the V regions of the Lchain and the H chain of the mouse antibody MABL-1, respectively, weremodified by the PCR method and introduced into the HEF expression vector(WO92/19759) to prepare vectors expressing chimera MABL-1 antibody.

A forward primer MLS (SEQ ID No. 9) for the L chain V region and aforward primer MHS (SEQ ID No. 10) for the H chain V region weredesigned to hybridize to a DNA encoding the beginning of the leadersequence of each V region and to contain the Kozak consensus sequence(J. Mol. Biol., 196, 947-950, 1987) and HindIII restriction enzyme site.A reverse primer MLAS (SEQ ID No. 11) for the L chain V region and areverse primer MHAS (SEQ ID No. 12) for the H chain V region weredesigned to hybridize to a DNA encoding the end of the J region and tocontain the splice donor sequence and BamHI restriction enzyme site.

100 μl of a PCR solution comprising 10 μl of 10×PCR Buffer II, 2 mMMgCl₂, 0.16 mM dNTPs (DATP, dGTP, dCTP and dTTP), 5 units of DNApolymerase AMPLITAQ GOLD, 0.4 μM each of primers and 8 ng of thetemplate DNA (pGEM-M1L or pGEM-M1H) was preheated at 94° C. of theinitial temperature for 9 minutes and then heated at 94° C. for 1minute, at 60° C. for 1 minute and at 72° C. for 1 minute 20 seconds inorder cycle was repeated 35 times and then the reaction mixture wasfurther heated at 72° C. for 10 minutes.

The PCR product was purified using the QIAquick PCR Purification Kit(QIAGEN) and then digested with HindIII and BamHI. The product from theL chain V region was cloned into the HEF expression vector, HEF-κ andthe product from the H chain V region was cloned into the HEF expressionvector, HEF-γ. After DNA sequencing, plasmids containing a DNA fragmentwith a correct DNA sequence are designated as HEF-M1L and HEF-M1H,respectively.

4.2 Preparation of Vectors Expressing Chimera MABL-2 Antibodies

Modification and cloning of cDNA were performed in the same mannerdescribed in Example 4.1 except for using pGEM-M2L and pGEM-M2H astemplate DNA instead of pGEM-M1L and pGEM-M1H. After DNA sequencing,plasmids containing DNA fragments with correct DNA sequences aredesignated as HEFM2L and HEF-M2H, respectively.

4.3 Transfection to COS7 Cells

The aforementioned expression vectors were tested in COS7 cells toobserve the transient expression of the chimera MABL-1 and MABL-2antibodies.

(1) Transfection with Genes for the Chimera MABL-1 Antibody

COS7 cells were co-transformed with the HEF-M1L and HEF-M1H vectors byelectroporation using the Gene Pulser apparatus (BioRad). Each DNA (10μg) and 0.8 ml of PBS with 1×10⁷ cells/ml were added to a cuvette. Themixture was treated with pulse at 1.5 kV, 25 μF of electric capacity.

After the restoration for 10 minutes at a room temperature, theelectroporated cells were transferred into DMEM culture medium (GIBCOBRL) containing 10% γ-globulinfree fetal bovine serum. After culturingfor 72 hours, the supernatant was collected, centrifuged to remove cellfragments and recovered.

(2) Transfection with Genes Coding for the Chimera MABL-2 Antibody

The co-transfection to COS7 cells with the genes coding for the chimeraMABL-2 antibody was carried out in the same manner as described inExample 4.3-(1) except for using the HEF-M2L and HEF-M2H vectors insteadof the HEF-M1L and HEF-MLH vectors. The supernatant was recovered in thesame manner.

4.4 Flow Cytometry

Flow cytometry was performed using the aforementioned culturesupernatant of COS7 cells to measure binding to the antigen. The culturesupernatant of the COS7 cells expressing the chimera MABL-1 antibody orthe COS7 cells expressing the chimera MABL-2 antibody, or human IgGantibody (SIGMA) as a control was added to 4×10⁵ cells of mouse leukemiacell line L1210 expressing human IAP and incubated on ice. Afterwashing, the FITC-labeled anti-human IgG antibody (Cappel) was addedthereto. After incubating and washing, the fluorescence intensitythereof was measured using the FACScan apparatus (BECTON DICKINSON).

Since the chimera MABL-1 and MABL-2 antibodies were specifically boundto L1210 cells expressing human IAP, it is confirmed that these chimeraantibodies have proper structures of the V regions of the mousemonoclonal antibodies MABL-1 and MABL-2, respectively (FIGS. 1-3).

Example 5 Preparation of Reconstructed Single chain Fv (scFv) of theAntibody MABL-1 and antibody MABL-2

5.1 Preparation of Reconstructed Single Chain Fv of Antibody MABL-1

The reconstructed single chain Fv of antibody MABL-1 was prepared asfollows. The H chain V region and the L chain V of antibody MABL-1, anda linker were respectively amplified by the PCR method and wereconnected to produce the reconstructed single chain Fv of antibodyMABL-1. The production method is illustrated in FIG. 4. Six primers(A-F) were employed for the production of the single chain Fv ofantibody MABL-1. Primers A, C and E have a sense sequence and primers B,D and F have an antisense sequence.

The forward primer VHS for the H chain V region (Primer A, SEQ ID No.13) was designed to hybridize to a DNA encoding the N-terminal of the Hchain V region and to contain NcoI restriction enzyme recognition site.The reverse primer VHAS for H chain V region (Primer B, SEQ ID No. 14)was designed to hybridize to a DNA coding the C-terminal of the H chainV region and to overlap with the linker.

The forward primer LS for the linker (Primer C, SEQ ID No. 15) wasdesigned to hybridize to a DNA encoding the N-terminal of the linker andto overlap with a DNA encoding the C-terminal of the H chain V region.The reverse primer LAS for the linker (Primer D, SEQ ID No. 16) wasdesigned to hybridize to a DNA encoding the C-terminal of the linker andto overlap with a DNA encoding the N-terminal of the L chain V region.

The forward primer VLS for the L chain V region (Primer E, SEQ ID No.17) was designed to hybridize to a DNA encoding the C-terminal of thelinker and to overlap with a DNA encoding the N-terminal of the L chainV region. The reverse primer VLAS-FLAG for L chain V region (Primer F,SEQ ID No. 18) was designed to hybridize to a DNA encoding theC-terminal of the L chain V region and to have a sequence encoding theFLAG peptide (Hopp. T. P. et al., Bio/Technology, 6, 1204-1210, 1988),two stop codons and EcoRI restriction enzyme recognition site.

In the first PCR step, three reactions, A-B, C-D and E-F, were carriedout and PCR products thereof were purified. Three PCR products obtainedfrom the first PCR step were assembled by their complementarity. Then,the primers A and F were added and the full length DNA encoding thereconstructed single chain Fv of antibody MABL-1 was amplified (SecondPCR). In the first PCR, the plasmid pGEM-M1H encoding the H chain Vregion of antibody MABL-1 (see Example 2), a plasmid pSC-DP1 whichcomprises a DNA sequence (SEQ ID NO: 19) encoding a linker regioncomprising: Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser(SEQ ID NO 118) (Huston, J. S., et al., Proc. Natl. Acad. Sci. USA, 85,5879-5883, 1988) and the plasmid pGEN-M1L encoding the L chain V regionof antibody MABL-I (see Example 2) were employed as template,respectively.

50 μl of the solution for the first PCR step comprises 5 p. 1 of 10×PCRBuffer II, 2 mM MgCl₂, 0.16 mM dNTPs, 2.5 units of DNA polymerase,AMPLITAQ GOLD (PERKIN ELMER), 0.4 μM each of primers and 5 ng each oftemplate DNA. The PCR solution was preheated at 94° C. of the initialtemperature for 9 minutes and then heated at 94° C. for 1 minute, at 65°C. for 1 minute and at 72° C. for 1 minute and 20 seconds in order. Thistemperature cycle was repeated 35 times and then the reaction mixturewas further heated at 72° C. for 7 minutes.

The PCR products A-B (371 bp), C-D (63 bp) and E-F (384 bp) werepurified using the QIAquick PCR Purification Kit (QIAGEN) and wereassembled in the second PCR. In the second PCR, 98 μl of a PCR solutioncomprising 120 ng of the first PCR product A-B, 20 ng of the PCR productC-D and 120 ng of the PCR product E-F, 10 μl of 10×PCR Buffer II, 2 mMMgCl₂, 0.16 mM dNTPs, 5 units of DNA polymerase AMPLITAQ GOLD (PERKINELMER) was preheated at 94° C. of the initial temperature for 8 minutesand then heated at 94° C. for 2 minutes, at 65° C. for 2 minutes and at72° C. for 2 minutes in order. This temperature cycle was repeated twiceand then 0.4 μM each of primers A and F were added into the reaction,respectively. The mixture was preheated at 94° C. of the initialtemperature for 1 minutes and then heated at 94° C. for 1 minute, at 65°C. for 1 minute and at 72° C. for 1 minute and 20 seconds in order. Thistemperature cycle was repeated 35 times and then the reaction mixturewas further heated at 72° C. for 7 minutes.

A DNA fragment of 843 by produced by the second PCR was purified anddigested by NcoI and EcoRI. The resultant DNA fragment was cloned intopSCFVT7 vector. The expression vector pSCFVT7 contains a pelB signalsequence suitable for E. coli periplasmic expression system (Lei, S. P.,et al., J. Bacteriology, 169, 4379-4383, 1987). After the DNAsequencing, the plasmaid containing the DNA fragment encoding correctamino acid sequence of the reconstructed single chain Fv of antibodyMABL-1 is designated as “pscMI” (see FIG. 5). The nucleotide sequence(SEQ ID NO: 20) and the amino acid sequence (SEQ ID NO: 119) of thereconstructed single chain Fv of antibody MABL-1 contained in theplasmid pscMI are shown in SEQ ID NO: 20).

The pscM1 vector was modified by the PCR method to prepare a vectorexpressing the reconstructed single chain Fv of antibody MABL-1 inmammalian cells. The resultant DNA fragment was introduced into pCHO1expression vector. This expression vector, pCHO1, was constructed bydigesting DHFR-ΔE-rvH-PM1-f (WO92/19759) with EcoRI and SmaI toeliminate the antibody gene and connecting the EcoRI-NotI-BamHI Adapter(Takara Shuzo) thereto.

As a forward primer for PCR, Sal-VHS primer shown in SEQ ID No. 21 wasdesigned to hybridize to a DNA encoding the N-terminal of the H chain Vregion and to contain SalI restriction enzyme recognition site. As areverse primer for PCR, FRH1 anti primer shown in SEQ ID No. 22 wasdesigned to hybridize to a DNA encoding the end of the first frameworksequence.

100 μl of PCR solution comprising 10 μl of 10×PCR Buffer II, 2 mM MgCl₂,0.16 mM dNTPs, 5 units of the DNA polymerase, AMPLITAQ GOLD, 0.4 μM eachof primer and 8 ng of the template DNA (pscM1) was preheated at 95° C.of the initial temperature for 9 minutes and then heated at 95° C. for 1minute, at 60° C. for 1 minute and at 72° C. for 1 minute and 20 secondsin order. This temperature cycle was repeated 35 times and then thereaction mixture was further heated at 72° C. for 7 minutes.

The PCR product was purified using the QIAquick PCR Purification Kit(QIAGEN) and digested by SalI and MbolI to obtain a DNA fragmentencoding the N-terminal of the reconstructed single chain Fv of antibodyMABL-1 The pscMI vector was digested by MbolI and EcoRI to obtain a DNAfragment encoding the C-terminal of the reconstructed single chain Fv ofantibody MABL-I. The SalI-MbolI DNA fragment and the MbolI-EcoRI DNAfragment were cloned into pCHO1-Igs vector. After DNA sequencing, theplasmid comprising the desired DNA sequence was designated as “pCHOMI”(see FIG. 6). The expression vector, pCHO1-Igs, contains a mouse IgGIsignal sequence suitable for the secretion-expression system inmammalian cells (Nature, 322, 323-327, 1988). The nucleotide sequence(SEQ ID NO: 23) and the amino acid-sequence (SEQ ID NO: 120) of thereconstructed single chain Fv of antibody MABL-I contained in theplasmid pCHOM1 are shown in SEQ ID NO 23.

5.2 Preparation of Reconstructed Single Chain Fv of Antibody MABL-2

The reconstructed single chain Fv of antibody MABL-2 was prepared inaccordance with the aforementioned Example 5.1. Employed in the firstPCR step were plasmid pGEN-N2H encoding the H chain V region of MABL-2(see Example 2) instead of pGEN-M1H and plasmid pGEM-M2L encoding the Lchain V region of MA.BL-2 (see Example 2) instead of pGEM-M1L, to obtaina plasmid pscM2 which comprises a DNA fragment encoding the desiredamino acid sequence of the single chain Fv of antibody MABL-2. Thenucleotide sequence (SEQ ID NO: 24) and the amino acid sequence (SEQ IDNO: 121) of the reconstructed single chain Fv of antibody MAEL-2contained in the plasmid pscM2 are shown in SEQ ID NO 24.

The pscM2 vector was modified by the PCR method to prepare a vector,pCHOM2, for the expression in mammalian cells which contains the DNAfragment encoding the correct amino acid sequence of reconstructed thesingle chain Fv, of antibody MABL-2. The nucleotide sequence (SEQ ID NO:25) and the amino acid sequence (SEQ ID NO: 122) of the reconstructedsingle chain Fv of antibody MABL-2 contained in the plasmid pCHOM2 areshown in SEQ ID NO 25.

5.3 Transfection to COS7 Cells

The pCHOM2 vector was tested in COS7 cells to observe the transientexpression of the reconstructed single chain Fv of antibody MABL-2.

The COS7 cells were transformed with the pCHOM2 vector byelectroporation using the Gene Pulser apparatus (BioRad). The DNA (10μg) and 0.8 ml of PBS with 1×10⁷ cells/ml were added to a cuvette. Themixture was treated with pulse at 1.5 kV, 25 μF of electric capacity.

After the restoration for 10 minutes at a room temperature, theelectroporated cells were transferred into IMDM culture medium (GIBCOBRL) containing 10% fetal bovine serum. After culturing for 72 hours,the supernatant was collected, centrifuged to remove cell fragments andrecovered.

5.4 Detection of the Reconstructed Single Chain Fv of Antibody MABL-2 inCulture Supernatant of COS7 Cells

The existence of the single chain Fv of antibody MABL-2 in the culturesupernatant of COS7 cells which had been transfected with the pCHOM2vector was confirmed by the Western Blotting method.

The culture supernatant of COS7 cells transfected with the pCHOM2 vectorand the culture supernatant of COS7 cells transfected with the pCHO1 asa control were subjected to SDS electrophoresis and transferred toREINFORCED NC membrane (Schleicher & Schuell). The membrane was blockedwith 5% skim milk (Morinaga Nyu-gyo), washed with 0.05% Tween 20-PBS andmixed with an ANTI-FLAG antibody (SIGMA). The membrane was incubated atroom temperature, washed and mixed with alkaline phosphatase-conjugatedmouse IgG antibody (Zymed). After incubating and washing at roomtemperature, the substrate solution (Kirkegaard Perry Laboratories) wasadded to develop color (FIG. 7).

A FLAG-peptide-specific protein was detected only in the culturesupernatant of the pCHOM2 vector-introduced COS7 cells and thus it isconfirmed that the reconstructed single chain Fv of antibody MABL-2 wassecreted in this culture supernatant.

5.5 Flow Cytometry

Flow cytometry was performed using the aforementioned COS7 cells culturesupernatant to measure the binding to the antigen. The culturesupernatant of the COS7 cells expressing the reconstructed single chainFv of antibody MABL-2 or the culture supernatant of COS7 cellstransformed with pCHO1 vector as a control was added to 2×10⁵ cells ofthe mouse leukemia cell line L1210 expressing human Integrin AssociatedProtein (IAP) or the cell line L1210 transformed with pCOS1 as acontrol. After incubating on ice and washing, the mouse ANTI-FLAGantibody (SIGMA) was added. Then the cells were incubated and washed.Then, the FITC labeled anti-mouse IgG antibody (BECTON DICKINSON) wasadded thereto and the cells were incubated and washed again.Subsequently, the fluorescence intensity was measured using the FACScanapparatus (BECTON DICKINSON).

Since the single chain Fv of antibody MABL-2 was specifically bound toL1210 cells expressing human IAP, it is confirmed that the reconstructedsingle chain Fv of antibody MABL-2 has an affinity to human IntegrinAssociated Protein (IAP) (see FIGS. 8-11).

5.6 Competitive ELISA

The binding activity of the reconstructed single chain Fv of antibodyMABL-2 was measured based on the inhibiting activity against the bindingof mouse monoclonal antibodies to the antigen.

The ANTI-FLAG antibody adjusted to 1 μg/ml was added to each well on96-well plate and incubated at 37° C. for 2 hours. After washing,blocking was performed with 1% BSA-PBS. After incubating and washing ata room temperature, the culture supernatant of COS7 cells into which thesecretion-type human IAP antigen gene (SEQ ID No. 26) had beenintroduced was diluted with PBS into twofold volume and added to eachwell. After incubating and washing at a room temperature, a mixture of500 of the biotinized MABL-2 antibody adjusted to 100 ng/ml and 50 p. 1of sequentially diluted supernatant of the COS7 cells expressing thereconstructed single chain Fv of antibody MABL-2 were added into eachwell. After incubating and washing at a room temperature, the alkalinephosphatase-conjugated streptoavidin (Zymed) was added into each well.After incubating and washing at a room temperature, the substratesolution (SIGMA) was added and absorbance of the reaction mixture ineach well was measured at 405 nm.

The results revealed that the reconstructed single chain Fv of antibodyMABL-2 (MABL2-scFv) evidently inhibited concentration-dependently thebinding of the mouse antibody MABL-2 to human IAP antigen in comparisonwith the culture supernatant of the PCHO1-introduced COS7 cells as acontrol. (FIG. 12). Accordingly, it is suggested that the reconstructedsingle chain Fv of antibody MABL-2 has the correct structure of each ofthe V regions from the mouse monoclonal antibody MABL-2.

5.7 Apoptosis-Inducing Effect In Vitro

An apoptosis-inducing action of the reconstructed single chain Fv ofantibody MABL-2 was examined by Annexin-V staining (Boehringer Mannheim)using the L1210 cells transfected with human IAP gene, the L1210 cellstransfected with the pCOS1 vector as a control and CCRF-CEM cells.

To each 1×10⁵ cells of the above cells was added the culture supernatantof the COS7 cells expressing the reconstructed single chain Fv ofantibody MABL-2 or the culture supernatant of COS7 cells transfectedwith the pCHO1 vector as a control at 50% final concentration and themixtures were cultured for 24 hours. Then, the Annexin-V staining wasperformed and the fluorescence intensity was measured using the FACScanapparatus (BECTON DICKINSON).

Results of the Annexin-V staining are shown in FIGS. 13-18,respectively. Dots in the left-lower region represent living cells anddots in the right-lower region represent cells at the early stage ofapoptosis and dots in the right-upper region represent cells at the latestage of apoptosis. The results show that the reconstructed single chainFv of antibody MABL-2 (MABL2-scFv) remarkably induced cell death ofL1210 cells specific to human IAP antigen (FIGS. 13-16) and that thereconstructed single chain Fv also induced remarkable cell death ofCCRF-CEM cells in comparison with the control (FIGS. 17-18).

5.8 Expression of MABL-2 Derived Single Chain Fv in CHO Cells

CHO cells were transfected with the pCHOM2 vector to establish a CHOcell line which constantly expresses the single chain Fv (polypeptide)derived from the antibody MABL-2.

CHO cells were transformed with the pCHOM2 vector by the electroporationusing the Gene Pulser apparatus (BioRad). A mixture of DNA (10 μg) and0.7 ml of PBS with CHO cells (1×10⁷ cells/ml) was added to a cuvette.The mixture was treated with pulse at 1.5 kV, 25 μF of electriccapacity. After the restoration for 10 minutes at a room temperature,the electroporated cells were transferred into nucleic acid free α-MEMmedium (GIBCO BRL) containing 10% fetal bovine serum and cultured. Theexpression of desired protein in the resultant clones was confirmed bySDS-PAGE and a clone with a high expression level was selected as a cellline producing the single chain Fv derived from the antibody MABL-2. Thecell line was cultured in serum-free medium CHO-S-SFM II (GIBCO BRL)containing 10 nM methotrexate (SIGMA). Then, the culture supernatant wascollected, centrifuged to remove cell fragments and recovered.

5.9 Purification of MABL-2 Derived Single Chain Fv Produced in CHO Cells

The culture supernatant of the CHO cell line expressing the single chainFv obtained in Example 5.8 was concentrated up to twenty times using acartridge for the artificial dialysis (PAN130SF, ASAHI MEDICALS). Theconcentrated solution was stored at −20° C. and thawed on purification.

Purification of the single chain Fv from the culture supernatant of theCHO cells was performed using three kinds of chromatography, i.e.,Blue-sepharose, a hydroxyapatite and a gel filtration.

(1) Blue-Sepharose Column Chromatography

The concentrated supernatant was diluted to ten times with 20 mM acetatebuffer (pH 6.0) and centrifuged to remove insoluble materials(10000×rpm, 30 minutes). The supernatant was applied onto aBlue-sepharose column (20 ml) equilibrated with the same buffer. Afterwashing the column with the same buffer, proteins adsorbed in the columnwere eluted by a stepwise gradient of NaCl in the same buffer, 0.1, 0.2,0.3, 0.5 and up to 1.0 M. The pass-through fraction and each elutedfraction were analyzed by SDS-PAGE. The fractions in which the singlechain Fv were confirmed (the fractions eluted at 0.1 to 0.3M NaCl) werepooled and concentrated up to approximately 20 times using CentriPrep-10(AMICON).

(2) Hydroxyapatite

The concentrated solution obtained in (1) was diluted to 10 times with10 mM phosphate buffer (pH 7.0) and applied onto the hydroxyapatitecolumn (20 ml, BIORAD). The column was washed with 60 ml of 10 mMphosphate buffer (pH 7.0). Then, proteins adsorbed in the column wereeluted by a linear gradient of sodium phosphate buffer up to 200 mM (seeFIG. 19). The analysis of each fraction by SDS-PAGE confirmed the singlechain Fv in fraction A and fraction B.

(3) Gel filtration

Each of fractions A and B in (2) was separately concentrated withCentriPrep-10 and applied onto TSKgel G3000SWG column (21.5×600 mm)equilibrated with 20 mM acetate buffer (pH 6.0) containing 0.15 M NaCl.Chromatograms are shown in FIG. 20. The analysis of the fractions bySDS-PAGE confirmed that both major peaks (AI and BI) are of desiredsingle chain Fv. In the gel filtration analysis, the fraction A waseluted at 36 kDa of apparent molecular weight and the fraction B waseluted at 76 kDa. The purified single chain Fvs (AI, BI) were analyzedwith 15% SDS polyacrylamide gel. Samples were treated in the absence orpresence of a reductant and the electrophoresis was carried out inaccordance with the Laemmli's method. Then the protein was stained withCoomassie Brilliant Blue. As shown in FIG. 21, both AI and BI gave asingle band at 35 kDa of apparent molecular weight, regardless of theabsence or presence of the reductant. From the above, it is concludedthat AI is a monomer of the single chain Fv and BI is a non-covalentlybound dimer of the single chain Fv. The gel filtration analysis of thefractions AI and BI with TSKgel G3000SW column (7.5×60 mm) revealed thata peak of the monomer is detected only in the fraction AI and a peak ofthe dimer is detected only in the fraction BI (FIG. 22). The dimerfraction (fraction BI) accounted for 4 period of total single chain Fvs.More than 90% of the dimer in the dimer fraction was stably preservedfor more than a month at 4° C.

5.10 Construction of Vector Expressing Single Chain Fv Derived fromAntibody MABL-2 in E. coli Cell

The pscM2 vector was modified by the PCR method to prepare a vectoreffectively expressing the single chain Fv from the antibody MABL-2 inE. coli cells. The resultant DNA fragment was introduced into pSCFVT7expression vector.

As a forward primer for PCR, Nde-VHSm02 primer shown in SEQ ID No. 27was designed to hybridize to a DNA encoding the N-terminal of the Hchain V region and to contain a start codon and NdeI restriction enzymerecognition site. As a reverse primer for PCR, VLAS primer shown in SEQID No. 28 was designed to hybridize to a DNA encoding the C-terminal ofthe L chain V region and to contain two stop codons and EcoRIrestriction enzyme recognition site. The forward primer, Nde-VHSm02,comprises five point mutations in the part hybridizing to the DNAencoding the N-terminal of the H chain V region for the effectiveexpression in E. coli.

100 μl of a PCR solution comprising 10 μl of 10×PCR Buffer #1, 1 mMMgCl₂, 0.2 mM dNTPs, 5 units of KOD DNA polymerase (all from TOYOBO), 1μM of each primer and 100 ng of a template DNA (pscM2) was heated at 98°C. for 15 seconds, at 65° C. for 2 seconds and at 74° C. for 30 secondsin order. This temperature cycle was repeated 25 times.

The PCR product was purified using the QIAquick PCR Purification Kit(QIAGEN) and digested by NdeI and EcoRI, and then the resulting DNAfragment was cloned into pSCFVT7 vector, from which pet B signalsequence had been eliminated by the digestion with NdeI and EcoRI. AfterDNA sequencing, the resulting plasmid comprising a DNA fragment with thedesired DNA sequence is designated as “pscM2Dem02” (see FIG. 23). Thenucleotide sequence (SEQ ID NO: 29) and the amino acid sequence (SEQ IDNO: 124) of the single chain Fv derived from the antibody MABL-2contained in the plasmid pscM2Dem02 are shown in SEQ ID NO 29.

5.11 Expression of Single Chain Fv Derived from Antibody MABL-2 in E.coli Cells

E. coli BL21(DE3)pLysS (STRATAGENE) was transformed with pscM2DEm02vector to obtain a strain of E. coli expressing the single chain Fvderived from antibody MABL-2. The resulting clones were examined for theexpression of the desired protein using SDS-PAGE, and a clone with ahigh expression level was selected as a strain producing the singlechain Fv derived from antibody MABL-2.

5.12 Purification of Single Chain Fv Derived from Antibody MABL-2Produced in E. coli

A single colony of E. coli obtained by the transformation was culturedin 3 ml of LB medium at 28° C. for 7 hours and then in 70 ml of LBmedium at 28° C. overnight. This pre-culture was transplanted to 7 L ofLB medium and cultured at 28° C. with stirring at 300-rpm using theJar-fermenter. When an absorbance of the medium reached O.D.=1.5, thebacteria were induced with 1 mM IPTG and then cultured for 3 hours.

The culture medium was centrifuged (10000×g, 10 minutes) and theprecipitated bacteria were recovered. To the bacteria was added 50 mMTris-HCl buffer (pH 8.0) containing 5 mM EDTA, 0.1 M NaCl and 1% TritonX-100 and the bacteria were disrupted by ultrasonication (out put: 4,duty cycle: 70%, 1 minute×10 times). The suspension of disruptedbacteria was centrifuged (12000×g, 10 minutes) to precipitate inclusionbody. Isolated inclusion body was mixed with 50 mM Tris-HCl buffer (pH8.0) containing 5 mM EDTA, 0.1 M NaCl and 4% Triton X-100, treated byultrasonication (out put: 4, duty cycle: 50%, 30 seconds×2 times) againand centrifuged (12000×g, 10 minutes) to isolate the desired protein asprecipitate and to remove containment proteins included in thesupernatant.

The inclusion body comprising the desired protein was lysed in 50 mMTris-HCl buffer (pH 8.0) containing 6 M Urea, 5 mM EDTA and 0.1 M NaCland applied onto Sephacryl S-300 gel filtration column (5×90 cm,Amersharm Pharmacia) equitibrated with 50 mM Tris-HCl buffer (pH 8.0)containing 4M Urea, 5 mM EDTA, 0.1 M NaCl and 10 mM mercaptoethanol at aflow rate of 5 ml/minutes to remove associated single chain Fvs withhigh-molecular weight. The obtained fractions were analyzed withSDS-PAGE and the fractions with high purity of the protein were dilutedwith the buffer used in the gel filtration up to O.D₂₈₀=0.25. Then, thefractions were dialyzed three times against 50 mM Tris-HCl buffer (pH8.0) containing 5 mM EDTA, 0.1 M NaCl, 0.5 M Arg, 2 mM glutathione inthe reduced form and 0.2 mM glutathione in the oxidized form in orderfor the protein to be refolded. Further, the fraction was dialyzed threetimes against 20 mM acetate buffer (pH 6.0) containing 0.15 M NaCl toexchange the buffer.

The dialysate product was applied onto Superdex 200 pg gel filtrationcolumn (2.6×60 cm, Amersharm Pharmacia) equilibrated with 20 mM acetatebuffer (pH 6.0) containing 0.15 M NaCl to remove a small amount of highmolecular weight protein which was intermolecularly crosslinked by S—Sbonds. As shown in FIG. 24, two peaks, major and sub peaks, were elutedafter broad peaks which are expectedly attributed to an aggregate with ahigh molecular weight. The analysis by SDS-PAGE (see FIG. 21) and theelution positions of the two peaks in the gel filtration analysissuggest that the major peak is of the monomer of the single chain Fv andthe sub peak is of the non-covalently bound dimer of the single chainFv. The non-covalently bound dimer accounted for 4 percent of totalsingle chain Fvs.

5.13 Apoptosis-Inducing Activity In Vitro of Single Chain Fv Derivedfrom Antibody MABL-2

An apoptosis-inducing action of the single chain Fv from antibody MABL-2(MABL2-scFv) produced by the CHO cells and E. coli was examinedaccording to two protocols by Annexin-V staining (Boehringer Mannheim)using the L1210 cells (hIAP/L1210) into which human IAP gene had beenintroduced.

In the first protocol sample antibodies at the final concentration of 3μg/ml were added to 5×10⁴ cells of hIAP/L1210 cell line and cultured for24 hours. Sample antibodies, i.e., the monomer and the dimer of thesingle chain Fv of MABL-2 from the CHO cells obtained in Example 5.9,the monomer and the dimer of the single chain Fv of MABL-2 from E. coliobtained in Example 5.12, and the mouse IgG antibody as a control wereanalyzed. After culturing., the Annexin-V staining was carried out andthe fluorescence intensity thereof was measured using the FACScanapparatus (BECTON DICKINSON).

In the second protocol sample antibodies at the final concentration of 3μg/ml were added to 5×10⁴ cells of hIAP/L1210 cell line, cultured for 2hours and mixed with ANTI-FLAG antibody (SIGMA) at the finalconcentration of 15 μg/ml and further cultured for 22 hours. Sampleantibodies of the monomer of the single chain Fv of MABL-2 from the CHOcells obtained in Example 5.9 and the mouse IgG antibody as a controlwere analyzed. After culturing, the Annexin-V staining was carriedout—and the fluorescence intensity thereof was measured using theFACScan apparatus.

Results of the analysis by the Annexin-V staining are shown in FIGS.25-31. The results show that the dimers of the single chain Fvpolypeptide of MABL-2 produced in the CHO cells and E. coli remarkablyinduced cell death (FIGS. 26, 27) in comparison with the control (FIG.25), while no apoptosis-inducing action was observed in the monomers ofthe single chain Fv polypeptide of MABL-2 produced in the CHO cells andE. coli (FIGS. 28, 29). When ANTI-FLAG antibody was used together, themonomer of the single chain Fv polypeptide derived from antibody MABL-2produced in the CHO cells induced remarkably cell death (FIG. 31) incomparison with the control (FIG. 30).

5.14 Antitumor Effect of the Monomer and the Dimer of scFv/CHOPolypeptide with a Model Mouse of Human Myeloma

(1) Quantitative Measurement of Human IgG in Mouse Serum

Measurement of human IgG (M protein) produced by human myeloma cell andcontained in mouse serum was carried out by the following ELISA. 100 μLof goat anti-human IgG antibody (BIOSOURCE, Lot#7902) diluted to 1 μg/mLwith 0.1% bicarbonate buffer (pH 9.6) was added to each well on 96 wellsplate (Nunc) and incubated at 4° C. overnight so that the antibody wasimmobilized. After blocking, 100 μL of the stepwisely diluted mouseserum or human IgG (CAPPEL, Lot#00915) as a standard was added to eachwell and incubated for 2 hours at a room temperature. After washing, 100μL of alkaline phosphatase-labeled anti-human IgG antibody (BIOSOURCE,Lot#6202) which had been diluted to 5000 times was added, and incubationwas carried out for 1 hour at a room temperature. After washing, asubstrate solution was added. After incubation, absorbance at 405 nm wasmeasured using the MICROPLATE READER Model 3550 (BioRad). Theconcentration of human IgG in the mouse serum was calculated based onthe calibration curve obtained from the absorbance values of human IgGas the standard.

(2) Preparation of Antibodies for Administration

The monomer and the dimer of the scFv/CHO polypeptide were respectivelydiluted to 0.4 mg/mL or 0.25 mg/mL with sterile filtered PBS(−) on theday of administration to prepare samples for the administration.

(3) Preparation of a Mouse Model of Human Myeloma

A mouse model of human myeloma was prepared as follows. KPMM2 cellspassaged in vivo (JP-Appl. 7-236475) by SCID mouse (Japan Clare) weresuspended in RPMI1640 medium (GIBCO-BRL) containing 10% fetal bovineserum (GIBCO-BRL) and adjusted to 3×10⁷ cells/mL. 200 μL of the KPMM2cell suspension (6×10⁶ cells/mouse) was transplanted to the SCID mouse(male, 6 week-old) via caudal vein thereof, which had beensubcutaneously injected with the asialo GM1 antibody (WAKO JUNYAKU, 1vial dissolved in 5 mL) a day before the transplantation.

(4) Administration of Antibodies

The samples of the antibodies prepared in (2), the monomer (250 μL) andthe dimer (400 μL), were administered to the model mice of human myelomaprepared in (3) via caudal vein thereof. The administration was startedfrom three days after the transplantation of KPMM2 cells and was carriedout twice a day for three days. As a control, 200 μL of sterile filteredPBS(−) was likewise administered twice a day for three days via caudalvein. Each group consisted of seven mice.

(5) Evaluation of Antitumor Effect of the Monomer and the Dimer ofscFv/CHO Polypeptide with the Model Mouse of Human Myeloma

The antitumor effect of the monomer and the dimer of scFv/CHOpolypeptide with the model mice of human myeloma was evaluated in termsof the change of human IgG (M protein) concentration in the mouse serumand survival time of the mice. The change of human IgG concentration wasdetermined by measuring it in the mouse serum collected at 24 days afterthe transplantation of KPMM2 cells by ELISA described in the above (1).The amount of serum human IgG (M protein) in the serum of thePBS(−)-administered group (control) increased to about 8500 μg/mL,whereas the amount of human IgG of the scFv/CHO dimer-administered groupwas remarkably low, that is, as low as one-tenth or less than that ofthe control group. Thus, the results show that the dimer of scFv/CHOstrongly inhibits the growth of the KPMM2 cells (FIG. 32). As shown inFIG. 33, a remarkable elongation of the survival time was observed inthe scFv/CHO dimer-administered group in comparison with thePBS(−)-administered group.

From the above, it is confirmed that the dimer of scFv/CHO has anantitumor effect for the human myeloma model mice. It is considered thatthe antitumor effect of the diner of scFv/CHO, the modified antibody ofthe invention, results from the apoptosis-inducing action of themodified antibody.

5.15 Hemagglutination Test

Hemagglutination test and determination of hemagglutination were carriedout in accordance with “Immuno-Biochemical Investigation”,Zoku-Seikagaku Jikken Koza, edited by the Biochemical Society of Japan,published by Tokyo Kagaku Dojin.

Blood was taken from a healthy donor using heparin-treated syringes andwashed with PBS(−) three times, and then erythrocyte suspension with afinal concentration of 2% in PBS(−) was prepared. Test samples were theantibody MABL-2, the monomer and the dimer of the single chain Fvpolypeptide produced by the CHO cells, and the monomer and the dimer ofthe single chain Fv polypeptide produced by E. coli, and the control wasmouse IgG (ZYMED). For the investigation of the hemagglutination effect,round bottom 96-well plates available from Falcon were used. 50 μL perwell of the aforementioned antibody samples and 50 μL of the 2%erythrocyte suspension were added and mixed in the well. Afterincubation for 2 hours at 37° C., the reaction mixtures were stored at4° C. overnight and the hemagglutination thereof was determined. As acontrol, 50 μL per well of PBS(−) was used and the hemagglutination testwas carried out in the same manner. The mouse IgG and antibody MABL-2were employed at 0.01, 0.1, 1.0, 10.0 or 100.0 μg/mL of the finalconcentration of the antibodies. The single chain Fvs were employed at0.004, 0.04, 0.4, 4.0, 40.0 or 80.0 μg/mL of the final concentration andfurther at 160.0 μg/mL only in the case of the dimer of the polypeptideproduced by E. coli. Results are shown in the Table 2. In the case ofantibody MABL-2, the hemagglutination was observed at a concentration ofmore than 0.1 μg/mL, whereas no hemagglutination was observed for boththe monomer and the dimer of the single chain Fv.

TABLE 2 H magglutination Test Control 0.01 0.1 1 10 100 μg/mL mlgG − − −− − − MABL-2 − − + +++ +++ ++ (intact) Control 0.004 0.04 0.4 4 40 80μg/mL scFv/CHO − − − − − − − monomer scFv/CHO − − − − − − − dimerControl 0.004 0.04 0.4 4 40 80 160 μg/mL scFv/E. coli − − − − − − −monomer scFv/E. coli − − − − − − − − dimer

Example 6 Modified Antibody sc(Fv)₂ comprising Two H Chain V Regions andTwo L Chain V Regions and Antibody MABL-2 scFvs Having Linkers withDifferent Length

6.1 Construction of Plasmid Expressing Antibody MABL-2 sc (Fv)₂

For the preparation of a plasmid expressing the modified antibody[sc(Fv)₂] which comprises two H chain V regions and two L chain Vregions derived from the antibody MABL-2, the aforementioned pCHOM2,which comprises the DNA encoding scFv derived from the MABL-2 describedabove, was modified by the PCR method as mentioned below and theresulting DNA fragment was introduced into pCHOM2.

Primers employed for the PCR are EF1 primer (SEQ ID NO: 30) as a senseprimer, which is designed to hybridize to a DNA encoding EF1α, and anantisense primer (SEQ ID NO: 19), which is designed to hybridize to theDNA encoding C-terminal of the L chain V region and to contain a DNAsequence coding for a linker region, and VLLAS primer containing SalIrestriction enzyme recognition site (SEQ ID NO 31).

100 μl of the PCR solution comprises 10 μl of 10×PCR Buffer #1, 1 mMMgCl₂, 0.2 mM dNTPs (DATP, dGTP, dCTP and dTTP), 5 units of KOD DNApolymerase (Toyobo, Inc.), 1 μM of each primer and 100 ng of thetemplate DNA (pCHOM2). The PCR solution was heated at 94° C. for 30seconds, at 50° C. for 30 seconds and at 74° C. for 1 minute in order.This temperature cycle was repeated 30 times.

The PCR product was purified using the QIAquick PCR Purification Kit(QIAGEN) and digested by SalI. The resultant DNA fragment was clonedinto pBluescript K5⁺ vector (Toyobo, Inc.). After DNA sequencing, aplasmid comprising the desired DNA sequence was digested by SalI and theobtained DNA fragment was conMected using Rapid DNA Ligation Kit(BOEHRINGER MANMHEIM) to pCHOM2 digested by SalI. After DNA sequencing,a plasmid comprising the desired DNA sequence is designated as“pCHOM2(Fv)₂” (see FIG. 34). The nucleotide sequence (SEQ ID NO: 32) andthe amino acid sequence (SEQ ID NO: 125) of the antibody MABL-2 sc(Fv)₂region contained in the plasmid pCHOM2(Fv)₂ are shown in SEQ ID NO 32.

6.2 Preparation of Plasmid Expressing Antibody MABL-2 scFvs HavingLinkers with Various Length

The scFvs containing linkers with different length and the V regionswhich are designed in the order of [H chain]-[L chain] (hereinafter“HL”) or [L chain]-[H chain] (hereinafter “LH”) were prepared using, asa template, cDNAs encoding the H chain and the L chain derived from theMABL-2 as mentioned below.

To construct HL type scFv the PCR procedure was carried out usingpCHOM2(Fv)₂ as a template. In the PCR step, a pair of CFHL-F1 primer(SEW ID NO: 33) and CFHL-R2 primer (SEQ ID NO: 34) or a pair of CFHL-F2primer (SEQ ID NO: 35) and CFHL-R1 primer (SEQ ID NO: 36) and KODpolymerase were employed. The PCR procedure was carried out by repeating30 times the temperature cycle consisting of 94° C. for 30 seconds, 60°C. for 30 seconds and 72° C. for 1 minute in order to produce a cDNA forthe H chain containing a leader sequence at 5′-end or a cDNA for the Lchain containing FLAG sequence at 3′-end thereof. The resultant cDNAsfor the H chain and the L chain were mixed and PCR was carried out byrepeating 5 times the temperature cycle consisting of 94° C. for 30seconds, 60° C. for 30 seconds and 72° C. for 1 minute in order usingthe mixture as templates and the KOD polymerase. To the reaction mixturewere added CFHL-F1 and CFHL-R1 primers and then the PCR reaction wasperformed by repeating 30 times of the aforementioned temperature cycleto produce a cDNA for HL-0 type without a linker.

To construct LH type scFv, the PCR reaction was carried out using, as atemplate, pGEM-M2L and pGEM-M2H which contain cDNAs encoding the L chainV region and the H chain V region from the antibody MABL-2, respectively(see JP-Appl. 11-63557). A pair of T7 primer (SEQ ID NO: 37) and CFLH-R2primer(SEQ ID NO: 38) or a pair of CFLH-F2 primer (SEQ ID NO: 39) andCFLH-R1 (SEQ ID NO: 40) and the KOD polymerase (Toyobo Inc.) wereemployed. The PCR reaction was performed by repeating 30 times thetemperature cycle consisting of 94° C. for 30 seconds, 60° C. for 30seconds and 72° C. for 1 minute in sequential order to produce a cDNA ofan L chain containing a leader sequence at 5′-end or a cDNA of an Hchain containing FLAG sequence at 3′-end thereof. The resultant cDNAs ofthe L chain and the H chain were mixed and PCR was carried out usingthis mixture as templates and the KOD polymerase by repeating 5 timesthe temperature cycle consisting of 94° C. for 30 seconds, 60° C. for 30seconds and 72° C. for 1 minute in order. To the reaction mixture wereadded T7 and CFLH-R1 primers and the reaction was performed by repeating30 times of the aforementioned temperature cycle. The reaction productwas used as a template and PCR was carried out using a pair of CFLH-F4primer (SEQ ID NO: 41) and CFLH-R1 primer by repeating 30 times thetemperature cycle consisting of 94° C. for 30 seconds, 60° C. for 30seconds and 72° C. for 1 minute in order to produce a cDNA of LH-0 typewithout a linker.

The resultant cDNAs of LH-0 and HL-0 types were digested by EcoRI andBamHI restriction enzymes (Takara Shuzo) and the digested cDNAs wereintroduced into an expression plasmid INPEP4 for mammalian cells usingLigation High (Toyobo Inc.), respectively. Competent E. coli JM109(Nippon Gene) was transformed with each plasmid and the desired plasmidswere isolated from the transformed E. coli using QIAGEN Plasmid Maxi Kit(QUIAGEN). Thus plasmids pCF2LH-0 and pCF2HL-0 were prepared.

To construct the expression plasmids of HL type containing linkers withdifferent size, pCF2HL-0, as a template, and CFHL-X3 (SEQ ID NO: 42),CFHL-X4 (SEQ ID NO: 43), CFHL-X5 (SEQ ID NO: 44), CFHL-X6 (SEQ ID NO:45) or CFHL-X7 (SEQ ID NO: 46), as a sense primer, and BGH-1 (SEQ ID NO:47) primer, as an antisense primer, which is complementary with thevector sequence were employed. PCR reaction was carried out using theKOD polymerase by repeating 30 times the temperature cycle consisting of94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute inorder and the reaction products were digested by restriction enzymesXhoI and BamHI (Takara Shuzo). The digested fragments were introducedbetween XhoI and BamHI sites in the pCF2HL-0 using Ligation High (ToyoboInc.), respectively. Competent E. coli JM109 was transformed with eachplasmid and the desired plasmids were isolated from the transformed E.coli by using Qiagen Plasmid Maxi kit. Thus expression plasmidspCF2HL-3, pCF2HL-4, pCF2HL-5, pCF2HL-6 and pCF2HL-7 were prepared.

To construct expression plasmid for the transient expression in COS7cells the plasmids pCF2HL-0, pCF2HL-3, pCF2HL-4, pCF2HL-5, pCF2HL-6 andpCF2HL-7 were digested by restriction enzymes EcoRI and BamHI (TakaraShuzo) and the resultant fragments of approximately 800 bp were purifiedwith agarose gel electrophoresis. The obtained fragments were introducedbetween EcoRI and BamHI sites in an expression plasmid pCOS1 for theexpression in mammalian cells by using Ligation High (Toyobo Inc.),respectively. Competent E. coli DH5α (Toyobo Inc.) was transformed witheach plasmid and the desired plasmids were isolated from the transformedE. coli using Qiagen Plasmid Maxi kit. Thus the expression plasmidsCF2HL-0/pCOS1, CF2HL-3/pCOS1, CF2HL4/pCOS1, CF2HL-5/pCOS1, CF2HL-6/pCOS1and CF2HL-7/pCOS1 were prepared.

As a typical example of these plasmids, the construction of the plasmidCF2HL-0/pCOS1 is illustrated in FIG. 35 and the nucleotide sequence (SEQID NO: 48) and the amino acid sequence (SEQ ID NO: 126) of MABL2-scFv<HL-0> contained in the plasmid are shown in SEQ ID NO 48. Nucleotidesequences and amino acid sequences (SEQ ID NOS 146-159, respectively, inorder of appearance) of the linker regions in these plasmids are alsoshown in FIG. 36.

To construct the expression plasmids of LH type containing linkers withdifferent size, pCF2LH-0, as a template, and CFLH-X3 (SEQ ID NO: 49),CFLH-X4 (SEQ ID NO: 50), CFLH-X5 (SEQ ID NO: 51), CFLH-X6 (SEQ ID NO:52) or CFLH-X7 (SEQ ID NO: 53), as a sense primer, and BGH-1 primer, asan antisense primer, which is complementary with the vector sequencewere employed. PCR reaction was carried out using the KOD polymerase byrepeating 30 times the temperature cycle consisting of 94° C. for 30seconds, 60° C. for 30 seconds and 72° C. for 1 minute in order and thereaction products were digested by restriction enzymes XhoI and BamHI.The digested fragments were introduced into the pCF2LH-0 between XhoIand BamHI sites using Ligation High, respectively. Competent E. coliDH5α (Toyobo Inc.) was transformed with each plasmid and the desiredplasmids were isolated from the transformed E. coli using Qiagen PlasmidMaxi kit. Thus expression plasmids pCF2LH-3, pCF2LH-4, pCF2LH-5,pCF2LH-6 and pCF2LH-7 were prepared.

To construct expression plasmid for the transient expression in COS7cells the plasmids pCF2LH-0, pCF2LH-3, pCF2LH-4, pCF2LH-5, pCF2LH-6 andpCF2LH-7 were digested by restriction enzymes EcoRI and BamHI (TakaraShuzo) and the resultant fragments of approximately 800 bp were purifiedwith agarose gel electrophoresis. The obtained fragments were introducedbetween XhoI and BamHI sites in an expression plasmid pCOS1 for theexpression in mammalian cells by using the Ligation High, respectively.Competent E. coli DH5α (Toyobo Inc.) was transformed with each plasmidand the desired plasmids were isolated from the transformed E. coliusing the Qiagen Plasmid Maxi kit. Consequently, the expression plasmidsCF2LH-0/pCOS1, CF2LH-3/pCOS1, CF2LH4/pCOS1, CF2LH-5/pCOS1, CF2LH-6/pCOS1and CF2LH-7/pCOS1 were prepared.

As a typical example of these plasmids, the construction of the plasmidCF2LH-0/pCOS1 is illustrated in FIG. 37 and the nucleotide sequence (SEQID NO: 54) and the amino acid (SEQ ID NO: 127) sequence of MABL2-scFv<LH-0> contained in the plasmid are shown in SEQ ID NO 54. Nucleotidesequences and amino acid sequences (SEQ ID NOS 160-173, respectively, inorder of appearance) of the linker regions in these plasmids are alsoshown in FIG. 38.

6.3 Expression of scFvs and sc(Fv)₂ in COS7 Cells

(1) Preparation of Culture Supernatant Using Serumcontaining CultureMedium

The HL type and LH type of scFvs and sc(Fv)₂ were transiently expressedin COS7 cells (JCRB9127, Japan Health Sciences Foundation). COS7 cellswere subcultured in DMEM media (GIBCO BRL) containing 10% fetal bovineserum (HyClone) at 37° C. in carbon dioxide atmosphere incubator. TheCOS7 cells were transfected with CF2HL-0, 3˜7/pCOS1, or CF2LH-0,3˜7/pCOS1 prepared in Example 6.2 or pCHOM2(Fv)₂ vectors byelectroporation using the Gene Pulser apparatus (BioRad). The DNA (10μg) and 0.25 ml of 2×10⁷ cells/ml in DMEM culture medium containing 10%FBS and 5 mM BES (SIGMA) were added to a cuvette. After standing for 10minutes the mixtures were treated with pulse at 0.17 kV, 950 μF ofelectric capacity. After the restoration for 10 minutes at roomtemperature, the electroporated cells were transferred into the DMEMculture medium (10% FBS) in 75 cm³ flask. After culturing for 72 hours,the culture supernatant was collected and centrifuged to remove cellfragments. The culture supernatant was subjected to the filtration using0.22 μm bottle top filter (FALCON) to obtain the culture supernatant(hereinafter “CM”).

(2) Preparation of Culture Supernatant Using Serum-Free Culture Medium

Cells transfected in the same manner as (1) were transferred to the DMEMmedium (10% FBS) in 75 cm³ flask and cultured overnight. After theculture, the supernatant was discarded and the cells were washed withPBS and then added to CHO-S-SFM II medium (GIBCO BRL). After culturingfor 72 hours, the culture supernatant was collected, centrifuged toremove cell fragments and filtered using 0.22 μm bottle top filter(FALCON) to obtain CM.

6.4 Detection of scFvs and sc(Fv)₂ in CM of COS7

The various MABL2-scFVs and sc(Fv)₂ in CM of COS7 prepared in theaforementioned Example 6.3 (2) were detected by Western Blotting method.

Each CM of COST was subjected to SDS-PAGE electrophoresis andtransferred to REINFORCED NC membrane (Schleicher & Schuell). Themembrane was blocked with 5% skim milk (Morinaga Nyu-gyo) and washedwith TBS. Then an ANTI-FLAG antibody (SIGMA) was added thereto. Themembrane was incubated at room temperature and washed. A peroxidaselabeled mouse IgG antibody (Jackson Immuno Research) was added. Afterincubating and washing at room temperature, the substrate solution(Kirkegaard Perry Laboratories) was added to develop color (FIG. 39).

6.5 Flow Cytometry

Flow cytometry was performed using the culture supernatants of COS7cells prepared in Example 6.3 (1) to measure the binding of theMABL2-scFVs and sc(Fv)₂ to human Integrin Associated Protein (IAP)antigen. The culture supernatants to be tested or a culture supernatantof COS7 cells as a control was added to 2×10⁵ cells of the mouseleukemia cell line L1210 expressing human IAP. After incubating on iceand washing, 10 μg/mL of the mouse ANTI-FLAG antibody (SIGMA) was addedand then the cells were incubated and washed. Then, the FITC labeledanti-mouse IgG antibody (BECTON DICKINSON) was added thereto and thecells were incubated and washed again. The fluorescence intensity wasmeasured using the FACScan apparatus (BECTON DICKINSON). The results ofthe flow cytometry show that the MABL2-scFvs having linkers withdifferent length and the sc(Fv)₂ in the culture supernatants of COS7have high affinity to human IAP (see FIGS. 40 a and 40b).

6.6 Apoptosis-Inducing Effect In Vitro

An apoptosis-inducing action of the culture supernatants of COS7prepared in Example 6.3 (1) was examined by Annexin-V staining(Boehringer Mannheim) using the L1210 cells transfected with human IAPgene (hIAP/L1210).

To 5×10⁴ cells of the hIAP/L1210 cells were added the culturesupernatants of COS7 cells transfected with each vectors or a culturesupernatant of COS7 cells as a control at 10% of the final concentrationand the mixtures were cultured for 24 hours. Then, the Annexin-V/PIstaining was performed and the fluorescence intensity was measured usingthe FACScan apparatus (BECTON DICKINSON). The results revealed thatscFvs <HL3, 4, 6, 7, LH3, 4, 6, 7> and sc(Fv)₂ in CM of COS7 inducedremarkable cell death of hIAP/L1210 cells. These results are shown inFIG. 41.

6.7 Construction of Vectors for the Expression of scFvs and sc(Fv)₂ inCHO Cells

To isolate and purify MABL2-scFvs and sc(Fv) 2 from culture supernatant,the expression vectors for expressing in CHO cells were constructed asbelow.

The EcoRI-BamHI fragments of pCF2HL-0, 3˜7, and pCF2LH-0, 3˜7 preparedin Example 6.2 were introduced between EcoRI and BamHI sites in anexpression vector pCHO1 for CHO cells using the Ligation High. CompetentE. coli DH5α was transformed with them. The plasmids were isolated fromthe transformed E. coli using QIAGEN Plasmid Midi kit (QIAGEN) toprepare expression plasmids pCHOM2HL-0, 3˜7, and pCHOM2LH-0, 3˜7.

6.8 Production of CHO Cells Expressing MABL2-scFvs <HL-0, 3˜7>,MABL2-scFvs <LH-0, 3˜7> and sc(Fv)₂ and Preparation of the CultureSupernatants Thereof

CHO cells were transformed with each of the expression plasmidspCHOM2HL-0, 3˜7, and pCHOM2LH-0, 3˜7, constructed in Example 6.7 andPCHOM2(Fv)₂ vector to prepare the CHO cells constantly expressing eachmodified antibody. As a typical example thereof, the production of theCHO cells constantly expressing MABL2-scFv <HL-5> or sc(Fv)₂ isillustrated as follows.

The expression plasmids pCHOM2HL-5 and pCHOM2(Fv)₂ were linearized bydigesting with a restriction enzyme PvuI and subjected to transfectionto CHO cells by electroporation using Gene Pulser apparatus (BioRad).The DNA (10 μg) and 0.75 ml of PBS with 1×10⁷ cells/ml were added to acuvette and treated with pulse at 1.5 kV, 25 μF of electric capacity.After the restoration for 10 minutes at room temperature, theelectroporated cells were transferred into nucleic acid-containing A-MEMculture medium (GIBCO BRL) containing 10% fetal bovine serum andcultured. After culturing overnight, the supernatant was discarded. Thecells were washed with PBS and added to nucleic acid-free A-MEM culturemedium (GIBCO BRL) containing 10% fetal bovine serum. After culturingfor two weeks, the cells were cultured in a medium containing 10 nM(final concentration) methotrexate (SIGMA), then 50 nM and 100 nMmethotrexate. The resultant cells were cultured in serum-free CHO-S-SFMII medium (GIBCO BRL) in a roller bottle. The culture supernatant wascollected, centrifuged to remove cell fragments and filtered using afilter with 0.22 μm of pore size to obtain CM, respectively.

According to the above, CHO cells which constantly express MABL2-scFvs<HL-0, -3, -4, -6, -7> and <LH-0, -3, -4, -5, -6, -7> and CMs thereofwere obtained.

6.9 Purification of Dimer of MABL2-scFv <HL-5> and sc(Fv)₂

The MABL2-scFv <HL-5> and the sc(Fv)₂ were purified from CMs prepared inExample 6.8 by two types of purification method as below.

<Purification Method 1>

HL-5 and sc(Fv)₂ were purified by the ANTI-FLAG antibody affinity columnchromatography utilizing the FLAG sequence located at C-terminal of thepolypeptides and by gel filtration. One liter of CM as obtained in 6.8was applied onto a column (7.9 ml) prepared with ANTI-FLAG M2 AFFINITYGEL (SIGMA) equilibrated with 50 mM Tris-HCl buffer (TBS, pH 7.5)containing 150 mM NaCl. After washing the column with TBS, the scFv waseluted by 0.1 M glycine-HCl buffer, pH 3.5. The resultant fractions wereanalyzed by SDS-PAGE and the elution of the scFv was confirmed. The scFvfraction was mixed with Tween 20 up to 0.01% of the final concentrationand concentrated using Centricon-10 (MILIPORE). The concentrate wasapplied onto TSKgel G3000SWG column (7.5×600 mm) equilibrated with 20 mMacetate buffer (pH 6.0) containing 150 mM NaCl and 0.01% Tween 20. At0.4 mL/minute of the flow rate, the scFv was detected by the absorptionat 280 nm. The HL-5 was eluted as the major fraction in the position ofthe dimer and the sc(Fv)₂ was eluted in the position of the monomer.

<Purification Method 2>

HL-5 and sc(Fv)₂ were purified using three steps comprising ion exchangechromatography, hydroxyapatite and gel filtration. In the ion exchangechromatography, Q sepharose fast flow column (Pharmacia) was employedfor HL-5 and SP-sepharose fast flow column was employed for sc(Fv)₂. Inand after the second step, HL-5 and sc(Fv)₂ were processed by the sameprocedure.

First Step for HL-5

CM of HL-5 was diluted to two times with 20 mM Tris-HCl buffer (pH 9.0)containing 0.02% Tween 20 and then the pH was adjusted to 9.0 with 1 MTris. The solution was applied onto Q Sepharose fast flow columnequilibrated with 20 mM Tris-HCl buffer (pH 8.5) containing 0.02% Tween20. A polypeptide adsorbed to the column was eluted by a linear gradientof NaCl in the same buffer, from 0.1 to 0.55 M. Monitoring the elutedfractions by SDS-PAGE, the fractions containing HL-5 were collected andsubjected to hydroxyapatite of the second step.

First Step for sc(Fv)₂

CM of the sc(Fv)₂ was diluted to two times with 20 mM acetate buffer (pH5.5) containing 0.02% Tween 20 and its pH was adjusted to 5.5 with 1 Macetic acid. The solution was applied onto a SP-Sepharpse fast flowcolumn equilibrated with 20 mM acetate buffer (pH 5.5) containing 0.02%Tween 20. A polypeptide adsorbed to the column was eluted by a lineargradient of NaCl in the buffer, from 0 to 0.5 M. Monitoring the elutedfractions by SDS-PAGE, the fractions containing the sc(Fv)₂ werecollected and subjected to hydroxyapatite of the second step.

Second step: Hydroxyapatite Chromatography of HL-5 and sc(Fv)₂

The fractions of HL-5 and sc(Fv)₂ obtained in the first step wereseparately applied onto the hydroxyapatite column (Type I, BIORAD)equilibrated with 10 mM phosphate buffer containing 0.02% Tween 20, pH7.0. After washing the column with the same buffer, polypeptidesadsorbed to the column were eluted by a linear gradient of the phosphatebuffer up to 0.5 M. Monitoring the eluted fractions by SDS-PAGE, thefractions containing the desired polypeptides were collected.

Third step: Gel Filtration of HL-5 and sc(Fv)₂

Each fraction obtained at the second step was separately concentratedwith CentriPrep-10 (MILIPORE) and applied onto a Superdex 200 column(2.6×60 cm, Pharmacia) equilibrated with 20 mM acetate buffer (pH 6.0)containing 0.02% Tween 20 and 0.15 M NaCl. HL-5 was eluted in theposition of the diner, and sc(Fv)HL-5- and sc(FV)₂ were eluted in theposition of the monomer as a major peek respectively.

Since the monomer of HL-5 was hardly detected by both purificationmethods, it is proved that the dimers of single chain Fvs are formed inhigh yields when the linker for the single chain Fv contains around 5amino acids. Furthermore, the dimer of HL-5 and the sc(Fv)₂ were stablypreserved for a month at 4° C. after the purification.

6.10 Evaluation of the Binding Activity of Purified Dimer of scFv <HL-5>and sc(Fv)₂ Against Antigen

Flow cytometry was performed using the purified dimer of MABL2-scFv<HL-5> and the purified sc(Fv)₂ in order to evaluate the binding tohuman Integrin Associated Protein (IAP) antigen. 10 μg/ml of thepurified dimer of MABL2-scFv <HL-5>, the purified sc(Fv)₂, the antibodyMABL-2 as a positive control or a mouse IgG (Zymed) as a negativecontrol was added to 2×10⁵ cells of the mouse leukemia cell line L1210expressing human IAP (hIAP/L1210) or the cell line L1210 transformedwith pCOS1 (pCOS1/L1210) as a control. After incubating on ice andwashing, 10 μg/mL of the mouse ANTI-FLAG antibody (SIGMA) was added andthen the cells were incubated and washed. FITC labeled anti-mouse IgGantibody (BECTON DICKINSON) was added thereto and the cells wereincubated and washed again. Then the fluorescence intensity was measuredusing the FACScan apparatus (BECTON DICKINSON).

Since the purified dimer of MABL2-scFv <HL-5> and the purified sc(Fv)₂were specifically bound to hIAP/L1210 cells, it is confirmed that thedimer of scFv <HL-5> and the sc(Fv)₂ have high affinity to human IAP(see FIG. 42).

6.11 Apoptosis-Inducing Activity In Vitro of Purified Dimer of scFv<HL-5> and sc(Fv)₂

An apoptosis-inducing action of the purified dimer of MABL2-scFv <HL-5>and the purified sc(Fv)₂ were examined by Annexin-V staining (BoehringerMannheim) using the L1210 cells (hIAP/L1210) in which human IAP gene hadbeen introduced and cells of human leukemic cell line CCRF-CEM.

Different concentrations of the purified dimer of MABL2-scFv <HL-5>, thepurified MABL2-sc(Fv)₂, the antibody MABL-2 as a positive control or amouse IgG as a negative control were added to 5×10⁴ cells of hIAP/L1210cell line or 1×10⁵ cells of CCRF-CEM cell line. After culturing for 24hours, the Annexin-V staining was carried out and the fluorescenceintensity thereof was measured using the FACScan apparatus (BECTONDICKINSON). As a result the dimer of MABL2-scFv <HL-5> and theMABL2-sc(Fv)₂ remarkably induced cell death of hHIAP/L1210 and CCRF-CEMin concentration-dependent manner (see FIG. 43). As a result it wasshown that the diner of MABL2-scFv <HL-5> and MABL2sc(Fv)₂, had improvedefficacy of inducing apoptosis compared with original antibody MABL-2.

6.12 Hemagglutination Test of the Purified Dimer of scFv <HL-5> and thesc(Fv)₂

Hemagglutination test was carried out using different concentrations ofthe purified diner of scFv <HL-5> and the purified sc(Fv)₂ in accordancewith Example 5.15.

The hemagglutination was observed with the antibody MABL-2 as a positivecontrol, whereas no hemagglutination was observed with both the singlechain antibody MABL2-sc(Fv)₂ and the MABL2-scFv <HL-5>. Further, therewas no substantial difference in the hemagglutination between twobuffers employed with the antibody MABL-2. These results are shown inTable 3.

TABLE 3 Hemagglutination Test Diluent: PBS (μg/ml) cont 28.9 14.45 7.2253.6125 1.8063 0.9031 0.4516 0.2258 MABL2- − − − − − − − − − sc(Fv)₂ cont28.0 14.0 7.0 3.5 1.75 0.875 0.4375 0.2188 MABL2- − − − − − − − − −sc(Fv) <HL5> cont 80 40 20 10 5 2.5 1.25 0.625 MABL2 − + + + + + + + +(intact) mlgG − − − − − − − − − Diluent: Acetate Buffer (μg/ml) cont 8040 20 10 5 2.5 1.25 0.625 MABL2 − + + + + + + + + (intact) Diluent: PBS(μg/ml) 0.1129 0.0564 0.0282 0.0141 0.0071 0.0035 0.0018 MABL2- − − − −− − − sc(Fv)₂ 0.1094 0.0547 0.0273 0.0137 0.0068 0.0034 0.0017 MABL2- −− − − − − − sc(Fv) <HL5> 0.3125 0.1563 0.0781 0.0391 0.0195 0.00980.0049 MABL2 + ± − − − − − (intact) mlgG − − − − − − − Diluent: AcetateBuffer (μg/ml) 0.3125 0.1563 0.0781 0.0391 0.0195 0.0098 0.0049MABL2 + + + − − − − (intact)6.13 Antitumor Effect of the Purified Dimer of scFv <HL-5> and thesc(Fv)₂ for a Model Mouse of Human Myeloma

The antitumor effects were tested for the dimer of scFv <HL-5> and thesc(Fv)₂ prepared and purified in Examples 6.8 and 6.9. The test wasperformed by using the mouse model for human myeloma produced in Example5.1 and determining the amount of M protein produced by human myelomacells in the mouse serum using ELISA and examining survival time of themice. Then, the antitumor effects of the dimer of scFv <HL-5> and thesc(Fv) 2 were evaluated in terms of the change of the amount of Mprotein in the mouse serum and the survival time of the mice.

In the test, the HL-5 and the sc(Fv)₂ were employed as a solution at0.01, 0.1 or 1 mg/mL in vehicle consisting of 150 mM NaCl, 0.02% Tweenand 20 mM acetate buffer, pH 6.0 and administered to the mice at 0.1, 1or 10 mg/kg of dosage. Control group of mice were administered only withthe vehicle.

The mouse serum was gathered 26 days after the transplantation of thehuman myeloma cells and the amount of M protein in the serum wasmeasured using ELISA according to Example 5.14. As a result, the amountof M protein in the serum of both mice groups administered with HL-5,the dimer and the sc(Fv)₂ decreased in dose-dependent manner (see FIG.44). Furthermore, a significant elongation of the survival time wasobserved in both groups administered with the HL-5 (FIG. 45) and withthe sc(Fv)₂ (FIG. 46) in comparison with the control group administeredwith the vehicle. These results show that the HL-5 and the sc(Fv)₂ ofthe invention have excellent antitumor effect in vivo.

Example 7 Single Chain Fv Comprising H Chain V Region and L Chain VRegion of Human Antibody 12B5 Against Human MPL

A DNA encoding V regions of human monoclonal antibody 12B5 against humanMPL was constructed as follows:

7.1 Construction of a Gene Encoding H Chain V Region of 12B5

The gene encoding H chain V region of human antibody 12B5 binding tohuman MPL was designed by conMecting the nucleotide sequence of the genethereof (SEQ ID NO: 55; encoded protein shown in SEQ ID NO: 128) at the5′-end to the leader sequence (SEQ ID NO: 56; encoded protein shown inSEQ ID NO: 129) originated from human antibody gene (Eur. J. Immunol.1996; 26: 63-69). The designed nucleotide sequence was divided into fouroligonucleotides having overlapping sequences of 15 by each (12B5VH-1,12B5VH-2, 12B5VH-3, 12B5VH-4). 12B5VH-1 (SEQ ID NO: 57) and 12B5VH-3(SEQ ID NO: 59) were synthesized in the sense direction, and 12BSVH-2(SEQ ID NO: 58) and 12B5VH-4 20 (SEQ ID NO: 60) in the antisensedirection, respectively. After assembling each synthesizedoligonucleotide by respective complementarity, the outside primers(12B5VH-S and 12B5VH-A) were added to amplify the full length of thegene. 12B5VH-S (SEQ ID NO: 61) was designed to hybridize to 5′-end ofthe leader sequence by the forward primer and to have Hind IIIrestriction enzyme recognition site and Kozak sequence, and 12B5VH-A(SEQ ID NO: 62) was designed to hybridize to the nucleotide sequenceencoding C-terminal of H chain V region by the reverse primer and tohave a splice donor sequence and BamHI restriction enzyme recognitionsite, respectively.

100 μl of the PCR solution containing 10 μl of 10×PCR GOLD BUFFER II,1.5 mM MgCl₂, 0.08 mM dNTPs (DATP, dGTP, dCTP, dTTP), 5 units ofDNA-polymerase AMPLITAQ GOLD (all by PERKIN ELMER) and each 2.5 p moleof each synthesized oligonucleotide (12B5VH-1 to −4) was heated at 94°C. of the initial temperature for 9 minutes, at 94° C. for 2 minutes, at55° C. for 2 minutes and 72° C. for 2 minutes. After repeating the cycletwo times each 100 pmole of external primer 12B5VH-S and 12B5VH-A wasadded. The mixture was subjected to the cycle consisting of at 94° C.for 30 seconds, at 55° C. for 30 seconds and 72° C. for 1 minute 35times and heated at 72° C. for further 5 minutes.

The PCR product was purified by 1.5% low-melting-temperature agarose gel(Sigma), digested by restriction enzymes BamHI and Hind III, and clonedinto expression vector HEF-gγ1 for human H chain. After determining theDNA sequence the plasmid containing the correct DNA sequence was namedHEF-12B5H-gγ1.

The HEF-12E10H-gγ1 was digested by restriction enzymes EcORI and BamHIto produce the gene encoding 12E10VH and then cloned into a human Fab Hchain expression vector pCOS-Fd to produce pFd-12E10H. The human Fab Hchain expression vector was constructed by amplifying the DNA (SEQ IDNO: 63; encoded protein shown in SEQ ID NO: 130) containing the intronregion existing between the genes encoding human antibody H chain Vregion and the constant region, and the gene encoding a part of thehuman H chain constant region by PCR, and inserting—the PCR productinto—animal cell expression vector pCOSb. The human H chain constantregion was amplified for the gene under the same conditions mentionedabove using as the template HEF-gγ1, as the forward primer G1CH1-S (SEQID NO: 64) which was designed to hybridize to 5′-end sequence of intron1 and to have restriction enzyme recognition sites EcoRI and BamHI andas the reverse primer G1CH1-A (SEQ ID NO: 65) which was designed tohybridize to 3′-end DNA of human H chain constant region CR1 domain andto have a sequence encoding a part of hinge region, two stop codons andrestriction enzyme recognition site 3g1 II.

The nucleotide sequence (SEQ ID NO: 66) and amino acid sequence (SEQ IDNO: 131) of the reconstructed 12E10 H chain variable region which wereincluded in plasmids HEF-I2EIOH-gγ1 and pFd-12E10H are shown in SEQ IDNO: 94.

7.2 Construction of the Gene Encoding 12B5 L Chain V Region

The gene encoding L chain V region of human antibody 12B5 binding tohuman MPL was designed by conMecting the nucleotide sequence of gene(SEQ ID NO: 67; encoded protein shown in SEQ ID NO: 132) at the 5′-endto the leader sequence (SEQ ID NO: 68; encoded protein shown in SEQ IDNO: 133) originated from human antibody gene 3D6 (Nuc. Acid Res. 1990:18; 4927). In the same way as mentioned above the designed nucleotidesequence was divided into four oligonucleotides having overlappingsequences of 15 by each (12B5VL-1, 12B5VL-2, 12B5VL-3, 12B5VL-4) andsynthesized respectively. 12B5VL-1 (SEQ ID NO: 69) and 12B5VL-3 (SEQ IDNO: 71) had sense sequences, and 12B5VL-2 (SEQ ID NO: 70) and 12B5VL-4(SEQ ID NO: 72) had antisense sequences, respectively. Each of thesynthesized oligonucleotides was assembled by respective complementarityand mixed with the external primer (12B5VL-S and 12B5VL-A) to amplifythe full length of the gene. 12B5VL-S (SEQ ID NO: 73) was designed tohybridize to 5′-end of the leader sequence by the forward primer and tohave Hind III restriction enzyme recognition site and Kozak sequence.12B5VL-A (SEQ ID NO: 74) was designed to hybridize to the nucleotidesequence encoding C-terminal of L chain V region by the reverse primerand to have a splice donor sequence and BamHI restriction enzymerecognition site.

Performing the PCR as mentioned above, the PCR product was purified by1.50 low-melting-temperature agarose gel (Sigma), digested byrestriction enzymes BamHI and Hind III, and cloned into an expressionvector HEF-gκ for human L chain. After determining the DNA sequence theplasmid containing the correct DNA sequence was named HEF-12B5L-gκ. Thenucleotide sequence (SEQ ID NO: 75) and amino acid sequence (SEQ ID NO:134) of the reconstructed 12B5 L chain V region which were included inplasmid HEF-12B5L-gκ are shown in SEQ ID NO: 75.

7.3 Production of Reconstructed 12B5 Single Chain Fv (scFv)

The reconstructed 12B5 antibody single chain Fv was designed to be inthe order of 12B5VH-linker-12B5VL and to have FLAG sequence (SEQ ID NO:76; encoded protein shown in SEQ ID NO: 135) at C-terminal to facilitatethe detection and purification. The reconstructed 12B5 single chain Fv(sc12B5) was constructed using a linker sequence consisting of 15 aminoacids represented by (Gly₄4Ser)₃(SEQ ID NO: 118).

(1) Production of the Reconstructed 12B5 Single Chain Fv Using theLinker Sequence Consisting of 15 Amino Acids

The gene encoding the reconstructed 12B5 antibody single chain Fv, whichcontained the linker sequence consisting of 15 amino acids, wasconstructed by connecting 12B5H chain V region, linker region and 12B5 Lchain V region which was amplified by PCR respectively. This method isschematically shown in FIG. 47. Six PCR primers (A-F) were used forproduction of the reconstructed 12B5 single chain Fv. Primers A, C, andE had sense sequences, and primers B, D, and F had antisense sequences.

The forward primer 12B5-S (Primer A, SEQ ID NO: 77) for H chain V regionwas designed to hybridize to 5′-end of H chain leader sequence and tohave EcoRI restriction enzyme recognition site. The reverse primerHuVHJ3 (Primer B, SEQ ID NO: 78) for H chain V region was designed tohybridize to DNA encoding C-terminal of H chain V region.

The forward primer RHuJH3 (Primer C, SEQ ID NO: 79) for the linker wasdesigned to hybridize to DNA encoding the N-terminal of the linker andto overlap DNA encoding the C-terminal of H chain V region. The reverseprimer RHuVK1 (Primer D, SEQ ID NO: 80) for the linker was designed tohybridize to DNA encoding the C-terminal of the linker and overlap DNAencoding the N-terminal of L chain V region.

The forward primer HuVK1.2 (Primer E, SEQ ID NO: 81) for L chain Vregion was designed to hybridize to DNA encoding the N-terminal of Lchain V region. The reverse primer 12B5F-A for L chain V region (PrimerF, SEQ ID NO: 82) was designed to hybridize to DNA encoding C-terminalof L chain V region and to have the sequence encoding FLAG peptide(Hopp, T. P. et al., Bio/Technology, 6, 1204-1210, 1988), twotranscription stop codons and NotI restriction enzyme recognition site.

In the first PCR step, three reactions A-B, C-D, and E-F were performed,and the three PCR products obtained from the first step PCR wereassembled by respective complementarity. After adding primers A and Fthe full length DNA encoding the reconstructed 12B5 single chain Fvhaving the linker consisting of 15 amino acids was amplified (the secondPCR). In the first step PCR, the plasmid HEF12B5H-g□l (see Example 7. 1)encoding the reconstructed 12B5 H chain V region, pSCFVT7-hM21(humanized ONS-M21 antibody) (Ohtomo et al., Anticancer Res. 18 (1998),4311-4316) containing DNA (SEQ ID NO: 83) encoding the linker regionconsisting of Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly GlySer (SEQ ID NO: 136) (Huston et al., Proc. Natl. Acad. Sci. USA, 85,5879-5883, 1988) and the plasmid HEF-12B5L-g□ (see Example 7. 2)encoding the reconstructed 12B5 L chain V region were used as templates,respectively.

50 μl of PCR solution for the first step contained 5 μl of 10×PCR GOLDBUFFER II, 1.5 mM MgCl₂, 0.08 mM dNTPs, 5 units of DNA polymeraseAMPLITAQ GOLD (all by PERKIN ELMER), each 100 pmole of each primer and10 ng of each template DNA. The PCR solution was heated at 94° C. of theinitial temperature for 9 minutes, at 94 for 30 seconds, 55° C. for 30seconds and 72° C. for 1 minute. After repeating the cycle 35 times thereaction mixture was further heated 72° C. for 5 minutes.

The PCR products A-B, C-D, and E-F were assembled by the second PCR. PCRmixture solution for the second step of 98 μl containing as the template1>1 of the first PCR product A-B, 0.5 μl of PCR product C-D and 1 μl ofPCR product E-F, 10 μl of 10×PCR GOLD BUFFER II, 1.5 mM MgCl₂, 0.08 mMdNTPs, 5 units of DNA polymerase AMPLITAQ GOLD (all by PERKIN ELMER) washeated at 94° C. of the initial temperature for 9 minutes, at 94° C. for2 minutes, at 65° C. for 2 minutes and 72° C. for 2 minutes. Afterrepeating the cycle two times, each 100 pmole of each of primers A and Fwere added. After repeating the cycle consisting of at 94° C. for 30seconds, 55° C. for 30 seconds and 72° C. for 1 minute 35 times, thereaction mixture was heated at 72° C. for 5 minutes.

The DNA fragments produced by the second PCR were purified using 1.5%low-melting-temperature agarose gel, digested by EcoRI and NotI, andcloned into pCHO1 vector and pCOS1 vector (Japanese Patent ApplicationNo. 8-255196). The expression vector pCHO1 was a vector constructed bydeleting the antibody gene from DHFR-ΔE-rvH-PM1-f (see WO92/19759) byEcoRI and SmaI digestion, and conMecting to EcoRI-NotI-BamHI Adaptor(TAKARA SHUZO). After determining the DNA sequence the plasmidscontaining the DNA fragment encoding the correct amino acid sequence ofreconstructed 12B5 single chain Fv⁻ were named pCHO-sc12B5 andpCOS-sc12B5. The nucleotide sequence (SEQ ID NO: 84) and amino acidsequence (SEQ ID NO: 137) of the Fv included in the plasmids shown inSEQ ID NO: 84.

7.4 Expression of Antibody 12B5 (IgG, Fab) and Single Chain FvPolypeptide by Animal Cell

Antibody 12B5 (IgG, Fab) and single chain Fv derived from antibody 12B5were expressed by using COS-7 cells or CHO cells.

The transient expression using COS-7 cells was performed as follows. Thetransfection was performed by electroporation method using Gene Pulserequipment (BioRad). For the expression of antibody 12B5 (IgG) each 10 μgof the above-mentioned expression vector HEF-12B5H-gγ1 and HEF-12 B5L-gKwere added, for the expression of 12B5Fab fragment each 10 μg ofpFd-12B5H and HEF-12B5L-gK were added and for the expression of singlechain Fv 10 μg of pCOS-sc12B5 was added to COS-7 cells (1×10⁷ cells/ml)suspended in 0.8 ml of PBS. The mixture kept in a cuvette was treated bypulse at the capacity of 1.5 kV, 25 μFD. After recovering for 10 minutesin a room temperature the electroporated cells were added to DMEMculture medium (GIBCO BRL) containing 10% bovine fetal serum cultivated.After cultivating overnight the cells were washed once by PBS, added toserum-free medium CHO-S-SFM II and cultivated for 2 days. The culturemedium was centrifuged to remove cell debris and filtered with 0.22 μmfilter to prepare the culture supernatant.

To establish a stable expression CHO cell line for the single chain Fv(polypeptide) derived from antibody 12B5, the expression vectorpCHO-sc12B5 was introduced into CHO cells as follows.

The expression vector was introduced into CHO cells by electroporationmethod using Gene Pulser equipment (BioRad). Linearized DNA (100 μg)obtained by digestion with restriction enzyme PvuI and CHO cells (1×10⁷cells/ml) suspended in 0.8 ml of PBS were mixed in a cuvette, leftstationary on ice for 10 minutes and treated with pulse at the capacityof 1.5 kV, 25 μFD. After recovering for 10 minutes at a room temperaturethe electroporated cells were added to CHO-S-SFM II (GIBCO BRL)containing 10% bovine fetal serum and cultivated. After cultivating for2 days the cultivation was continued in CHO-S-SFM II (GIBCO BRL)containing 5 nM methotrexate (SIGMA) and 10% bovine fetal serum. Fromthus obtained clones a clone with high expression rate was selected asthe production cell line for 12B5 single chain Fv. After cultivating inserum-free medium CHO-S-SFM II (GIBCO BRL) containing 5 nM methotrexate(SIGMA), the culture supernatant was obtained by centrifugal separationof cell debris.

7.5 Purification of Single Chain Fv Derived from 12B5 Produced by CHOCells

The culture supernatant of CHO cell line expressing 12B5 single chain Fvobtained in 7.4 was purified by ANTI-FLAG antibody column and gelfiltration column.

(1) ANTI-FLAG Antibody Column

The culture supernatant was added to ANTI-FLAG anti FLAG M2 AFFINITY GEL(SIGMA) equilibrated by PBS. After washing the column by the same bufferthe proteins adsorbed to the column were eluted by 0.1M glycine-HClbuffer (pH 3.5). The eluted fractions were immediately neutralized byadding 1M Tris-HCl buffer (pH 8.0). The eluted fractions were analyzedby SDS-PAGE and the fraction which was confirmed to contain the singlechain Fv was concentrated using Centricon-10 (MILLIPORE).

(2) Gel Filtration

The concentrated solution obtained in (1) was added to Superdex200column (110×300 mm, AMERSHAM PHARMACIA) equilibrated by PBS containing0.01% Tween20. The product sc12B5 was eluted in two peaks (A, B) (seeFIG. 48). The fractions A and B were analyzed using the14%-SDSpolyacrylamide gel. The sample was processed by electrophoresisin the presence and absence of a reducing agent according to Laemmlimethod, and stained by Coomassie Brilliant Blue after theelectrophoresis. As shown in FIG. 49 the fractions A and B, regardlessof the presence of the reducing agent or its absence, produced a singleband having an apparent molecular weight of about 31 kD. When thefractions A and B were analyzed by gel filtration using Superdex200 PC3.2/30 (3.2×300 mm, AMERSHAM PHARMACIA), the fraction A produced aneluted product at an apparent molecular weight of about 44 kD and thefraction B produced at 22 kD (see FIGS. 50 a and b). The results showthat the fraction A is the non-covalent bond dimer of sc12B5 singlechain Fv, and B is the monomer.

7.6 Measurement of TPO-Like Agonist Activity of Various Single Chain Fvs

The TPO-like activity of anti-MPL single chain antibody was evaluated bymeasuring the proliferation activity to Ba/F3 cells (BaF/mpl) expressinghuman TPO receptor (MPL). After washing BaF/Mpl cells two times byRPMI1640 culture medium (GIBCO) containing 10% bovine fetal serum(GIBCO), the cells were suspended in the culture medium at cell densityof 5×10⁵ cells/ml. The anti-MPL single chain antibody and human TPO(R&DSystems) was diluted with the culture medium, respectively. 50 μl of thecell suspension and 50 μl of the diluted antibody or human TPO wereadded in 96-well microplate (flat bottom) (Falcon), and cultivated inCO₂ incubator (CO₂ concentration: 5%) for 24 hours. After the incubation10 μl of WST-8 reagent (reagent for measuring the number of raw cellsSF: Nacalai Tesque) was added and the absorbance was immediatelymeasured at measurement wavelength of 450 nm and at reference wavelengthof 620 nm using fluorescence absorbency photometer SPECTRA Fluor(TECAN). After incubating in CO₂ incubator (CO₂ concentration: 5%) for 2hours, the absorbance at 450 nm of measurement wavelength and 620 nm ofreference wavelength was again measured using SPECTRA Fluor. Since WST-8reagent developed the color reaction depending upon the number of livecells at wavelength of 450 nm, the proliferation activity of BaF/Mplbased on the change of absorbance in 2 hours was evaluated by ED 50calculated as follows. In the proliferation reaction curve wherein theabsorbance was plotted on the ordinate against the antibodyconcentration on the abscissa, the absorbance at the plateau was set100% reaction rate. Obtaining an approximation formula by straight lineapproximation method based on the plotted values close to 50% reactionrate, the antibody concentration of 50% reaction rate was calculated andadopted as ED 50.

The results of the agonist activity to MPL measured by using culturesupernatants of COS-7 cells expressing various 12B5 antibody moleculesshowed as illustrated in FIG. 51 that 12B5IgG having bivalentantigen-binding site increased the absorbance in concentration-dependentmanner and had TPO-like agonist activity (ED50; 29 nM), while theagonist activity of 12B5Fab having monovalent antigen-biding site wasvery weak (ED50; 34, 724 nM). On the contrary the single chain Fv(sc12B5) having monovalent antigen-binding site like Fab showed strongagonist activity at a level that ED50 was 75 nM. However it has beenknown that variable regions of H chain and L chain of the single chainFv are associated through non-covalent bond and, therefore, eachvariable region is dissociated in a solution and can be associated withvariable region of other molecule to form multimers like diners. Whenthe molecular weight of sc12B5 purified by gel filtration was measured,it was confirmed that that there were molecules recognized to be monomerand diner (see FIG. 48). Then monomer sc12B5 and diner sc12B5 wereisolated (see FIG. 50) and measured for the agonist activity to MPL. Asshown in FIGS. 51 and 52, ED50 of sc12B5 monomer was 4438.7 nM, whichconfirmed that the agonist activity was reduced compared with the resultusing culture supernatant of COS-7 cells. On the contrary single chainFv (sc12B5 diner) having bivalent antigen-binding site showed about400-fold stronger agonist activity (ED50; 10.1 nM) compared withmonovalent sc12B5. Furthermore, the bivalent single chain Fv showed theagonist activity equivalent to or higher than the agonist activity ofhuman TPO and 12B5IgG.

Example 8 Construction of a Gene Encoding the Variable Region of HumanAntibody 12E10 Against Human MPL

A DNA encoding variable region of human monoclonal antibody 12E10against human MPL was constructed as follows:

8.1 Construction of a Gene Encoding 12E10H Chain V Region

The nucleotide sequence SEQ ID NO:86 was designed as a gene encoding Hchain V region of human antibody 12E10 binding to human MPL on the basisof the amino acid sequence described in WO99/b0494 (SEQ ID NO:85). Thefull length of nucleotide sequence was designed by conMecting to its5′-end the leader sequence (SEQ ID NO:87; encoded protein shown in SEQID NO:138) derived from human antibody gene (GenBank accession No.AF062252). The designed ucleotide sequence was divided into fouroligonucleotides having overlapping sequences of 15 by each (12E10VH1,12E10VH2, 12E10VH3, 12E10VH4), 12E10VH1 (SEQ ID NO: 88) and 12E10VH3(SEQ ID NO: 90) were synthesized in the sense direction, and 12E10VH2(SEQ ID NO: 89) and 12E10VH4 (SEQ ID NO: 91) in the antisense direction,respectively. After assembling each synthesized oligonucleotide byrespective complementarity, the external primers (12E10VHS and 12E10VHA)were added to amplify the full length of the gene. 12E10VHS (SEQ ID NO:92) was designed to hybridize to 5′-end of the leader sequence by theforward primer and to have Hind III restriction enzyme recognition siteand Kozak sequence, and 12E10VHA (SEQ ID NO: 93) was designed tohybridize to the nucleotide sequence encoding C-terminal of H chain Vregion by the reverse primer and to have a splice donor sequence andBam.HI restriction enzyme recognition site, respectively.

100 μl of the PCR solution containing 10 μl of 10×PCR GOLD BUFFER II,1.5 mM MgCl₂, 0.08 nM dNTPs (DATP, dGTP, dCTP, dTTP), 5 units ofDNA-polymerase AMPLITAQ GOLD (all by PERKIN ELMER) and each 2.5 pmole ofeach synthesized oligonucleotide (12B5VH-1 to −4) was heated at 94° C.of the initial temperature for 9 minutes, at 94° C. for 2 minutes, at55° C. for 2 minutes and 72° C. for 2 minutes. After repeating the cycletwo times each 100 pmole of external primer 12E10VHS and 12E10VHA wereadded. The mixture was subjected to the cycle consisting of at 94° C.for 30 seconds, at 55° C. for 30 seconds and 72° C. for 1 minute 35times and heated at 72° C. for further 5 minutes.

The PCR product was purified by 1.5% low-melting-temperature agarose gel(Sigma), digested by restriction enzymes BamHI and Hind III, and clonedinto a human H chain expression vector HEF-gγ1. After determining theDNA sequence the plasmid containing the correct DNA sequence was namedHEF-12E10H-gγ1.

The HEF-12E10H-gγ1 was digested by restriction enzymes EcoRI and BamHIto produce the gene encoding 12E10VH and then cloned into a human Fab Hchain expression vector pCOS-Fd to produce pFd-12E10H. The human Fab Hchain expression vector was constructed by amplifying the DNA (SEQ IDNO: 63) containing the intron region existing between the genes encodinghuman antibody H chain V region and the constant region, and the geneencoding a part of the human H chain constant region by PCR, andinserting the PCR product into animal cell expression vector pCOS1. Thehuman H chain constant region was amplified for the gene under the sameconditions mentioned above using as the template HEF-gγ1, as the forwardprimer G1CH1-S (SEQ ID NO: 64) which was designed to hybridize to 5′-endsequence of intron 1 and to have restriction enzyme recognition sitesEcoRI and BamHI and as the reverse primer G1CH1-A (SEQ ID NO: 65) whichwas designed to hybridize to 3′-end DNA of human H chain constant regionCH1 domain and to have a sequence encoding a part of hinge region, twostop codons and restriction enzyme recognition site Bg1 II.

The nucleotide sequence (SEQ ID NO: 94) and amino acid sequence (SEQ IDNO: 139) of the reconstructed 12E10 H chain variable region which wereincluded in plasmids HEF-I2EIOH-gγ1 and pFd-12E10H are shown in SEQ IDNO: 94.

8.2 Construction of a Gene Encoding 12E10 L Chain V Region

The nucleotide sequence SEQ ID NO:96 was designed as a gene encoding Lchain V region of human antibody 12E10 binding to human MPL on the basisof the amino acid sequence described in WO99/10494 (SEQ ID NO:95). Itwas further designed by conMecting to its 5′-end the leader sequence(SEQ ID NO: 97; encoded protein shown in SEQ ID NO: 140) derived fromhuman antibody gene (Mol. Immunol. 1992; 29: 1515-1518). In the same wayas mentioned above the designed nucleotide sequence was divided intofour oligonucleotides having overlapping sequences of 15 by each(12E10VL1, 12E10VL2, 12E10VL3, 12E10VL4) and synthesized respectively.12E10VL1 (SEQ ID NO: 98) and 12E10VL3 (SEQ ID NO: 100) had sensesequences, and 12E10VL2 (SEQ ID NO: 99) and 12E10VL4 (SEQ ID NO: 101)had antisense sequences, respectively. Each of the synthesizedoligonucleotides was assembled by respective complementarity and mixedwith the external primers (12E10VLS and 12E10VLA) to amplify the fulllength of the gene. 12E10VLS (SEQ ID NO: 102) was designed to hybridizeto 5′-end of the leader sequence by the forward primer and to have EcoRIrestriction enzyme recognition site and Kozak sequence. 12E10VLA (SEQ IDNO: 103) was designed to hybridize to the nucleotide sequence encodingC-terminal of L chain V region by the reverse primer and to have a B1nIrestriction enzyme recognition site.

Performing the PCR as mentioned above, the PCR product was purified by1.5% low-melting-temperature agarose gel (Sigma), digested byrestriction enzymes EcoRI and BinI, and cloned into pUCI9 containing agene for human lambda chain constant region. After determining the DNAsequence the plasmid containing the correct DNA sequence was digested byEcoRI to produce a gene encoding 12E10L chain V region and human lambdachain constant region and then inserted in expression vector pCOS1. Theplasmid having 12E10L chain gene (SEQ ID NO: 104; encoded protein shownin SEQ ID NO: 141) was named pCOS-I2EIOL.

8.3 Production of Reconstructed 12E10 Single Chain Fv

The reconstructed 12E10 antibody single chain Fv was designed to be inthe order of 12E10VH-linker-12E10VL and to have FLAG sequence (SEQ IDNO: 105;

-   -   encoded protein shown in SEQ ID NO: 142) at C-terminal to        facilitate the detection and purification. The reconstructed        b2E10 chain Fvs (sci2EbO and dbI2EIO) were constructed using a        linker sequence consisting of 15 amino acids represented by        (Gly₄Ser)₃ (SEQ ID NO: 118) or 5 amino acids represented by        (Gly₄Ser)₁ (SEQ ID NO: 176). (1) Production of the reconstructed        12E10 single chain Fv using the linker sequence consisting of 5        amino acids.

The gene encoding the reconstructed 12E10 single chain Fv, whichcontained the linker sequence consisting of 5 amino acids, wasconstructed by introducing the nucleotide sequence for the linker(Gly₄Ser)₁ (SEQ ID NO:176) to 3′-end of the gene encoding 12E10 H chainV region and to 5′-end of the gene encoding 12E10L chain V region,amplifying thus obtained respective gene by PCR and conMecting theamplified genes. Four PCR primers (A-D) were used to produce thereconstructed 12E10 single chain Fv. Primers A and C had sensesequences, and primers B and D—had antisense sequences.

The forward primer for H chain V region was I2EIOS (Primer A, SEQ ID NO:106). The reverse primer DB2 (Primer ‘B, SEQ ID NO: 107) for H chain Vregion was designed to hybridize to DNA encoding C-terminal of H chain Vregion and to have the nucleotide sequence encoding the linker(Gly₄Ser)₁ (SEQ ID NO: 176) and the nucleotide sequence encodingN-terminal of L chain V region.

The forward primer DB1 (Primer C, SEQ ID NO: 108) for L chain V regionwas designed to hybridize to DNA encoding the N-terminal of L chain Vregion and to have the nucleotide sequence encoding the linker(Gly₄Ser)₁ (SEQ ID NO: 176) and the nucleotide sequence encodingC-terminal of H chain V region. The reverse primer 12E10FA (Primer D,SEQ ID NO: 109) for L chain V region was designed to hybridize to DNAencoding the C-terminal of L chain V region and to have the nucleotidesequence encoding FLAG and NotI restriction enzyme recognition site.

In the first PCR step, two reactions A-B and C-D were performed, and thetwo PCR products obtained from the first step PCR were assembled byrespective complementarity. After adding primers A and D the full lengthDNA encoding the reconstructed 12E10 single chain Fv having the linkerconsisting of 5 amino acids was amplified (the second PCR). In the firststep PCR, the plasmid HEF-12E10H-Gyl (see Example 8.2) encoding thereconstructed 12E10 H chain V region and pCOS-12E10L (see Example 8.1)encoding the reconstructed 12E10L chain V region were used as templates,respectively.

50 μl of the first step PCR solution contained 5 μl of 10×PCR GOLDBUFFER II, 1.5 mM MgCl₂, 0.8 mM dNTPs, 5 units of DNA polymeraseAMPLITAQ GOLD (by PERKIN ELMER), each 100 pmole of each primer and 100ng of each template DNA. The PCR solution was heated at 94° C. of theinitial temperature for 9 minutes, at 94 for 30 seconds, 55° C. for 30seconds and 72° C. for 1 minute. After repeating the cycle 35 times thereaction mixture was further heated at 72° C. for 5 minutes.

The PCR products A-B (429 bp) and C-D (395 bp) were assembled by thesecond PCR. The second step PCR mixture solution (9811) containing 1 μleach of the first PCR product A-B and C-D as templates, 100 pmole eachof each primer, 10 μl of 10×PCR GOLD BUFFER II, 1.5 mM MgCl₂, 0.08 mMdNTPs and 5 units of DNA polymerase AMPLITAQ GOLD (by PERKIN ELMER) wasreacted under the same conditions as mentioned above.

The DNA fragment of 795 bp produced by the second PCR was purified using1.5% low-melting-temperature agarose gel, digested by EcoRI and NotI,and cloned into pCHO1 vector or pCOS1 vector. The expression vectorpCHO1 was a vector constructed by deleting the antibody gene fromDHFR-ΔE-RVH-PM1-f (see w092119759) by EcoRI and SmaI digestion, andconMecting to EcoRI-NotI-BamHI Adaptor (TAKARA SHUZO) After determiningthe DNA sequence the plasmids containing the DNA fragment encoding thecorrect amino acid sequence of reconstructed 12B5 single chain Fv werenamed pCHO-dbI2EIO and pCOS-dbI2EIO. The nucleotide sequence (SEQ ID NO:110) and amino acid sequence (SEQ ID NO: 143) of the reconstructed I2EIOsingle chain Fv included in the plasmids pCHO-dbI2EIO and pCOS-dbI2EIOare shown in SEQ ID NO: 110.

(2) Production of the Reconstructed 12E10 Single Chain Fv Using theLinker Sequence Consisting of 15 Amino Acids

The gene encoding the reconstructed I2EIO antibody single chain Fv,which contained the linker sequence consisting of 15 amino acids, wasconstructed by introducing the nucleotide sequence for the linker(Gly₄Ser)₃ (SEQ ID NO: 118) to 3′-end of the gene encoding b2E10 H chainV region and to 5′-end of the gene encoding 12EIOL chain V region,amplifying thus obtained respective gene by PCR and conMecting theamplified genes. Four PCR primers (A-D) were used for production of thereconstructed I2EIO single chain Fv. Primers A and C had sensesequences, and primers B and D had antisense sequences.

The forward primer for H chain V region was 12EIO S (Primer A, SEQ IDNO: 106). The reverse primer sc4.3 (Primer 3, SEQ ID NO: 111) for Hchain V region was designed to hybridize to DNA encoding C-terminal of Hchain V region and to have the nucleotide sequence encoding the linker(Gly₄Ser)₃ (SEQ ID NO: 118) and the nucleotide sequence encodingN-terminal of L chain V region.

The forward primer scl.3 (Primer C, SEQ ID NO: 112) for L chain V regionwas designed to hybridize to DNA encoding the N-terminal of L chain Vregion and to have the nucleotide sequence encoding the linker(Gly₄Ser)₃ (SEQ ID NO: 118) and the nucleotide sequence encodingC-terminal of H chain V region. The reverse primer 12E10FA (Primer D,SEQ ID NO: 109) for L chain V region was designed to hybridize to DNAencoding the C-terminal of L chain V region and to have the nucleotidesequence encoding FLAG and NotI restriction enzyme recognition site.

In the first PCR step, two reactions A-B and C-D were performed, and thetwo PCR products obtained from the first step PCR were assembled byrespective complementarity. After adding primers A and D the full lengthDNA encoding the reconstructed 12E10 single chain Fv having the linkerconsisting of 15 amino acids was amplified (the second PCR). In thefirst step PCR, the plasmid pCOS-db12E10 (see Example 8.3(1)) encodingthe reconstructed 12E10 single chain Fv was used as template.

50 μl of the first step PCR solution contained 5 μl of 10×ExTaq Buffer,0.4 mM dNTPs, 2.5 units of DNA polymerase TaKaRa ExTaq (by TAKARA), each100 pmole of each primer and 10 ng of each template DNA. The PCRsolution was heated at 94° C. of the initial temperature for 30 seconds,at 94 for 15 seconds and 72° C. for 2 minute, and the cycle was repeated5 times. After repeating 28 times the cycle of at 94° C. for 15 secondsand at 70° C. for 2 minutes, the reaction mixture was further heated at72° C. for 5 minutes.

The PCR products A-B (477 bp) and C-D (447 bp) were assembled by thesecond PCR. The second step PCR mixture solution (98 μl) containing 1 μleach of the first PCR products A-B and C-D as templates, 100 pmole eachof each primer A and D, 5 μl of 10×ExTaq Buffer, 0.4 mM dNTPs, 2.5 unitsof DNA polymerase TaKaRa ExTaq (by TAKARA) was reacted under the sameconditions as mentioned above.

The DNA fragment of 825 bp produced by the second PCR was purified using1.0% low-melting-temperature agarose gel, digested by EcoRI and NotI.Thus obtained DNA fragment was cloned into pCHO1 vector or pCOS1 vector.After determining the DNA sequence the plasmids containing the DNAfragment encoding the correct amino acid sequence of reconstructed i2EIOsingle chain Fv were named pCHO-sc12EIO and pCOSscI2EIO. The nucleotidesequence (SEQ ID NO: 113) and amino acid sequence (SEQ ID NO: 144) ofthe reconstructed 12E10 single chain Fv included in the plasmidspCHO-scI2EIO and pCOS-scI2EIO are shown in SEQ ID NO: 113.

8.4 Expression of Antibody 12E10 (IgG, Fab) and Single Chain FvPolypeptide by Animal Cell

Antibody 12E10 (IgG, Fab) and single chain Fv derived from antibody12E10 (linker sequence 5 amino acids, 15 amino acids) were expressed byusing COS-7 cells or CHO cells.

The transient expression using COS-7 cells was performed as follows. Thetransfection was performed by electroporation method using Gene PulserII equipment (BioRad). For the expression of antibody 12E10 (IgG) each10 μg of the above-mentioned expression vector HEF-12E10H-gγ1 andpCOS-12E10L were added, for the expression of 12E10Fab fragment each 10μg of pFd-12E10H and pCOS-12E10L were added and for the expression ofsingle chain Fv of pCOS-sc12E10 (10 μg) or pCOS-db12E10 (10 μg) wasadded to COS-7 cells (1×10⁷ cells/ml) suspended in 0.8 ml of PBS. Themixture kept in a cuvette was treated by pulse at the capacity of 1.5kV, 25 μFD. After recovering for 10 minutes in a room temperature theelectroporated cells were added to DMEM medium (GIBCO BRL) containing10% bovine fetal serum and cultivated. After cultivating overnight thecells were washed once by PBS, added to serum-free medium CHO-S-SFM II(GIBCO BRL) and cultivated for 3 days. The culture supernatant wascentrifuged to remove cell debris and filtered with 0.22 μm filter.

To establish a stable expression CHO cell line for the single chain Fv(polypeptide) derived from antibody 12E10, the expression vectorpCHO-sc12E10 or pCHO-dsl2E10 was introduced into CHO cells respectively.

Each expression vector was introduced into CHO cells by electroporationmethod using Gene Pulser II equipment (BioRad). Linearized DNA (100 μg)obtained by digestion with restriction enzyme PvuI and CHO cells (1×10⁷cells/ml) suspended in 0.8 ml of PBS were mixed in a cuvette, leftstationary on ice for 10 minutes and treated with pulse at the capacityof 1.5 kV, 25 μFD. After recovering for 10 minutes at a room temperaturethe electroporated cells were added to CHO-S-SFM II medium (GIBCO BRL)containing 10% dialyzed bovine fetal serum and nucleic acid andcultivated. After cultivating for 2 days the cultivation was continuedin nucleic acid-free CHO-S-SFM II medium (GIBCO BRL) containing 10%dialyzed bovine fetal serum. From thus obtained clones a clone with highexpression rate was selected as the production cell line for 12E10single chain Fv. After cultivating in serum-free CHO-S-SFM II medium(GIBCO BRL), the culture supernatant was centrifuged to remove celldebris and filtered with 0.22 μm filter.

8.5 Purification of Single Chain Fv Derived from 12E10 Produced by CHOCells

The culture supernatants produced by CHO cell lines expressing 12E10single chain Fvs (sc12E10, db12E10) obtained in Example 8.4 werepurified by ANTI-FLAG antibody column and gel filtration columnrespectively to produce purified single chain Fvs.

(1) Purification with ANTI-FLAG Antibody Column

Each culture supernatant (sc12E10, db12E10) was added to ANTI-FLAG M2AFFINITY GEL column (SIGMA) equilibrated by 50 mM Tris-HCl buffer(pH7.4) containing 150 mM NaCl. After washing the column by the samebuffer the proteins adsorbed to the column were eluted by 100 mM glycinebuffer (pH 3.5). The eluted fractions were immediately neutralized byadding 1M Tris-HCl buffer (pH 8.0) and analyzed by SDS-PAGE. Thefraction which was confirmed to contain the single chain Fv was pooledand concentrated about 20-fold using Centricon-10 (AMICON).

(2) Gel Filtration

The concentrated solution obtained in (1) was added to Superdex200column HR (10×300 mm, AMERSHAM PHARMACIA) equilibrated by PBS containing0.01% Tween20. Chlomatograms were shown in FIGS. 53 and 54. The productsc12E10 was eluted in two peaks (A, B) (see FIG. 53). The productdb12E10 was eluted in two peaks (C, D) (see FIG. 54). Each peak fractionwas collected, treated in the presence and absence of a reducing agent,processed by electrophoresis according to Laemmli method and stained byCoomassie Brilliant Blue after the electrophoresis. As shown in FIG. 55the all of fractions A, B, C and D, regardless of the presence orabsence of the reducing agent, produced a single band having an apparentmolecular weight of about 31 kD. When these fractions were analyzed bygel filtration using Superdex200 HR, the fraction A produced a producteluted at an apparent molecular weight of about 2 kD, the fraction B at20 kD (see FIG. 56), fraction C at 69 kD and fraction D at 41 kD (seesFIG. 57). The results suggest that sc12E10-derived fraction A is thenon-covalent bond dimer of single chain fV and the fraction B is themonomer of single chain Fv, and the db12E10-derived fraction C is thenon-covalent bond trimer of single chain Fv and D is non-covalent bonddimer of single chain Fv.

8.6 Measurement of TPO-Like Agonist Activity of Various Single Chain Fvs

The TPO-like activity of anti-mpl single chain antibody was evaluated bymeasuring the proliferation activity to Ba/F3 cells (BaF/mpl) expressinghuman TPO receptor (MPL).

After washing BaF/mpl cells two times by RPMI1640 medium (GIBCO)containing 1% bovine fetal serum (GIBCO), the cells were suspended inthe medium at cell density of 5×10⁵ cells/mL. The anti-MPL single chainantibody or human TPO (R&D Systems) was diluted with the medium,respectively. 50 μl of the cell suspension and 50 μl of the dilutedantibody or human TPO were added in 96-well microplate (flat bottom)(Corning), and cultivated in CO₂ incubator (CO₂ concentration: 5%) for24 hours. After the incubation 10 μl of WST-8 reagent (reagent formeasuring the number of raw cells SF: Nacalai Tesque) was added and theabsorbance was immediately measured at measurement wavelength of 450 nmand at reference wavelength of 655 nm using absorbency photometerBenchmark Plus (BioRad). After incubating in CO₂ incubator (CO₂concentration: 5%) for 2 hours, the absorbance at 450 nm of measurementwavelength and 655 nm of reference wavelength was again measured usingBenchmark Plus. Since WST-8 reagent developed the color reactiondepending upon the number of live cells at wavelength of 450 nm, theproliferation activity of BaF/mpl was evaluated based on the change ofabsorbance in 2 hours.

The agonist activity to MPL measured by using culture supernatants ofCOS-7 cells expressing various 12E10 antibody molecules are shown inFIG. 58. Single chain Fvs having the 5-amino-acid-linker (dsl2E10) andthe 15-amino-acid-linker (sc12E10) increased the absorbance inconcentration-dependent manner, showing TPO-like agonist activity (ED50;9 pM and 51 pM respectively), while 12E10IgG and 12E10Fab had noactivity.

It has been known that H chain and L chain of the single chain Fv areassociated not only within a molecule but also between molecules to formmultimers such as dimer. When the culture supernatants of CHO cellsexpressing single chain Fvs of 12E10 were gel filtrated and tested foragonist activity on MPL. The results were shown in FIG. 59. The diner,which was contained in sc12E10 in a small amount, showed about 5000-foldstronger TPO-like agonist activity (sc12E10 diner, ED50; 1.9 pM)compared with the monomer (sc12E10 monomer, ED50; > 10 nM). The activitywas higher than that of TPO (ED50; 27 pM). The diner of db12E10 (db12E10dimer, ED50; 2.0 pM) showed strong activity comparable to that ofsc12E10 dimer db12E10 trimer (ED50; 7.4 pM), which was presumed to be atrimer from molecular weight obtained by gel filtration, showed a highactivity which is lower than that of db12E10 dimer. Those resultssuggest that it is important for the activity of agonist antibody 12E10that the antigen-binding site is bivalent (dimer). Considering the factthat 12E10 IgG had no activity, other factors than being bivalent arepresumed to be important such as the location of antigen-binding site,the distance or the angle.

INDUSTRIAL APPLICABILITY

The modified antibodies of the invention have an agonist action capableof transducing a signal into cells by crosslinking a cell surfacemolecule(s) and are advantageous in that the permeability to tissues andtumors is high due to the lowered molecular size compared with antibodymolecule (whole IgG). This invention provides the modified antibodieswith an agonist activity remarkably higher than TPO or parent antibodies(whole IgG). Especially even parent antibodies without agonist activitycan be altered into the modified antibodies with an agonist activityhigher than TPO. Therefore the modified antibodies can be used assignal-transducing agonists. The modification of antibody moleculeresults in the reduction of side effects caused by intercellularcrosslinking and provides novel medicines inducing only required actionby crosslinking a cell surface molecule(s). Medical preparationscontaining as active ingredient the modified antibodies of the inventionare useful as preventives and/or remedies for platelet-related-blooddiseases, thrombocytopenia caused by chemotherapy for cancers orleukemia and the like.

1. A modified antibody comprising two H chain V regions and two L chainV regions of an antibody, showing TPO agonist action by crosslinking TPOreceptors of the same cell, wherein: said modified antibody is a dimerof single chain Fv comprising one H chain V region and one L chain Vregion, wherein the H chain V region and L chain V region are connectedthrough a peptide linker having 15 amino acids, at least one of the Hchain V regions comprises a polypeptide having a sequence encoded by SEQID NO. 66 or SEQ ID NO. 94, and/or at least one of the L chain V regionscomprises a polypeptide having a sequence encoded by SEQ ID NO. 75 orSEQ ID NO. 104, and said modified antibody has a TPO agonist actionequivalent to or greater than that of thrombopoietin.
 2. The modifiedantibody of claim 1, wherein the modified antibody further comprises anamino acid sequence(s) for peptide purification.
 3. The modifiedantibody of claim 1, wherein the modified antibody has been purified. 4.The modified antibody of claim 1, wherein the modified antibody is amono-specific modified antibody.
 5. The modified antibody of claim 1,wherein the modified antibody is a multi-specific modified antibody. 6.The modified antibody of claim 5, wherein the modified antibody is abi-specific modified antibody.
 7. The modified antibody of claim 1,wherein the two L chain V regions and the two H chain V regions are fromthe same monoclonal antibody.
 8. The modified antibody of claim 1 whichsubstantially has no intercellular adhesion action.
 9. A medicinecomprising as active ingredient the modified antibody of claim
 1. 10.The medicine of claim 9 which is for the treatment of thrombocytopenia.11. A modified antibody comprising two H chain V regions and two L chainV regions of an antibody, showing TPO agonist action by crosslinking TPOreceptors of the same cell, wherein: said modified antibody is a dimerof a single chain FIT comprising one H chain V region and one L chain Vregion, at least one of the H chain V regions comprises a polypeptidehaving a sequence encoded by SEQ ID NO. 66 or SEQ ID NO. 94, and/or atleast one of the L chain V regions comprises a polypeptide having asequence encoded by SEQ ID NO. 75 or SEQ ID NO. 104, said modifiedantibody has a TPO agonist action equivalent to or greater than that ofthrombopoietin, and the peptide linker that connects the H chain Vregion and L chain V region is selected from the group consisting of:Gly-Gly-Ser Ser-Gly-Gly Gly-Gly-Gly-Ser  (SEQ ID NO: 174)Ser-Gly-Gly-Gly  (SEQ ID NO: 175) Gly-Gly-Gly-Gly-Ser  (SEQ ID NO: 176)Ser-Gly-Gly-Gly-Gly  (SEQ ID NO: 177) Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 178) Ser-Gly-Gly-Gly-Gly-Gly  (SEQ ID NO: 179)Gly-Gly-Gly--Gly-Gly-Gly-Ser  (SEQ ID NO: 180)Ser-Gly-Gly-Gly-Gly-Gly-Gly. (SEQ ID NO: 181)